Bismuth-thiols as antiseptics for epithelial tissues, acute and chronic wounds, bacterial biofilms and other indications

ABSTRACT

Compositions and methods, including novel homogeneous microparticulate suspensions, are described for treating acute wounds, chronic wounds and/or a wound or epithelial tissue surface that contains bacterial biofilm, including unexpected synergy between bismuth-thiol (BT) compounds and certain antibiotics, to provide topical formulations including antiseptic formulations, for management and promotion of wound healing and in particular infected wounds. Previously unpredicted antibacterial properties and anti-biofilm properties of disclosed BT compounds and BT compound-plus-antibiotic combinations are also described, including preferential efficacies of certain such compositions for treating gram-positive bacterial infections, and distinct preferential efficacies of certain such compositions for treating gram-negative bacterial infections.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 U.S.C. 119(e) of U.S.Provisional Patent Application No. 61/149,593 filed Feb. 3, 2009, whichis incorporated herein by reference in its entirety.

BACKGROUND Technical Field

The presently disclosed invention embodiments relate to compositions andmethods for the treatment of microbial infections. In particular, thepresent embodiments relate to improved treatments for managing bacterialinfections in epithelial tissues, including in wounds such as chronicwounds and acute wounds, and including treatment of bacterial biofilmsand other conditions.

Description of the Related Art

The complex series of coordinated cellular and molecular interactionsthat contribute to skin wound healing, and/or to healing or maintenanceof epithelial tissues generally, may be adversely impacted by a varietyof external factors, such as opportunistic and nosocomial infections(e.g., clinical regimens that can increase the risk of infection), localor systemic administration of antibiotics (which may influence cellgrowth, migration or other functions and can also select forantibiotic-resistant microbes), frequent wound dressing changes,open-air exposure of wounds to speed healing, the use of temporaryartificial structural support matrix or scaffold materials, and/or thepossible need for debridement and/or repeat surgery to excise infectedor necrotic tissue.

Wound healing thus continues to be a formidable challenge for clinicalpractitioners worldwide. The current treatments for recalcitrant woundsare impractical and ineffective, often requiring multiple surgeries toclose the wound. For instance, Regranex® (becaplermin, Ortho-McNeilPharmaceutical, Inc., available from Ethicon, Inc., recombinantplatelet-derived growth factor) exemplifies one of the few availabletreatments for chronic wounds, but is expensive to produce and haslimited clinical utility.

Chronic and Acute Wounds and Wound Biofilms

Wounds occur when the continuity between cells within a tissue, orbetween tissues, is disrupted, for instance, by physical, mechanical,biological, pathological and/or chemical forces (e.g., burns, dermalinfections, puncture wounds, gunshot or shrapnel wounds, skin ulcers,radiation poisoning, malignancies, gangrene, autoimmune disease,immunodeficiency disease, respiratory insult such as by inhalation orinfection, gastrointestinal insult such as by deleterious ingestion orinfection, circulatory and hematologic disorders including clottingdefects,) or other traumatic injuries, or the like.

While a limited level of bacterial contamination in a wound, or“colonization” of the wound, may not necessarily interfere with theprocesses of wound healing, the presence of bacteria in numberssufficient to overwhelm the host immune defenses can lead to an acutewound or a chronic wound or a wound in which a bacterial biofilm ispresent, such as a wound infection in which bacterial growth proceeds tothe detriment of the host. Bryant and Nix, Acute and Chronic Wounds:Current Management Concepts, 2006 Mosby (Elsevier), NY; Baronoski, WoundCare Essentials: Practical Principles (2^(nd) Ed.), 2007 Lippincott,Williams and Wilkins, Philadelphia, Pa.). For example, acute wounds suchas may result from injury, trauma, surgical intervention, or othercauses, typically lack underlying health deficits and heal rapidly, butmay on occasion fail to do so due to the presence of an infection;rapidly forming bacterial biofilms have been described in acute wounds(e.g., WO/2007/061942). Additional factors that may contribute to thedevelopment of chronic wounds include losses in mobility (e.g., thatresult in continued pressure being applied to a wound site), deficits ofsensation or mental ability, inaccessibility of the wound site (e.g., inthe respiratory or gastrointestinal tracts) and circulatory deficits.Infection at a chronic wound site may be detected by the clinical signsof skin redness, edema, pus formation and/or unpleasant odor, or otherrelevant, clinically accepted criteria.

Acute wounds that cannot heal properly may thus be present, and chronicwounds thus may develop, in higher organisms (including but not limitedto humans and other mammals) when the host's immune system has beenoverwhelmed by bacterial infection of a wound site (e.g., an acutewound), creating permissive conditions for bacteria to invade andfurther destroy tissue. In general, chronic wounds are wounds that donot heal within three months, and instead of becoming smaller they tendto grow larger as the bacterial infiltration progresses. Chronic woundsmay become very painful and stressful for the patient when nearby nervesbecome damaged (neuropathy) as the wound progresses. These wounds affectfour million Americans each year and cost about $9 billion in treatmentexpenses. Afflicted individuals are mostly over the age of 60.

Chronic wounds may in some cases originate as acute wounds and thus mayinclude, for example, gunshot or shrapnel wounds, burns, punctures,venous ulcers, pressure ulcers, diabetic ulcers, radiation poisoning,malignancies, dermal infections, gangrene, surgical wounds, diabeticfoot ulcers, decubitis ulcers, venous leg ulcers, infected and/orbiofilm-containing nonhealing surgical wounds, pyoderma gangrenosum,traumatic wounds, acute arterial insufficiency, necrotizing fasciitis,osteomyelitis (bone infection), and radiation injuries, such asosteoradionecrosis and soft tissue radionecrosis, or other types ofwounds. Venous ulcers, for example, occur mostly in the legs, as aresult of poor circulation (e.g., ischemia), malfunctioning valves ofveins, or repeated physical trauma (e.g., repetitive injury). Pressureulcers may be present when local pressure that is exerted at or around awound site is greater than blood pressure, for instance, such that poorcirculation, paralysis, and/or bed sores may contribute to, orexacerbate, the chronic wound. Diabetic ulcers may occur in individualswith diabetes mellitus, for example, persons in whom uncontrolled highblood sugar can contribute to a loss of feeling in the extremities,leading to repetitive injuries and/or neglect on the part of theindividual to attend to injuries. Factors that can complicate orotherwise influence clinical onset and outcome of chronic wounds includethe subject's immunological status (e.g., immune suppression,pathologically (e.g., HIV-AIDS), radiotherapeutically orpharmacologically compromised immune system; age; stress); skin aging(including photochemical aging), and development and progression ofbiofilms within the wound. In the case of epithelial tissues in therespiratory and/or gastrointestinal tracts, inaccessibility, occlusion,difficulty in generating epithelial surface-clearing fluid forces ordevelopment of localized microenvironments conducive to microbialsurvival can engender clinical complications.

Wound-related injuries may be accompanied by lost or compromised organfunction, shock, bleeding and/or thrombosis, cell death (e.g., necrosisand/or apoptosis), stress and/or microbial infection. Any or all ofthese events, and especially infection, can delay or prevent theeffective tissue repair processes that are involved in wound healing.Hence, it can be important as early as possible in an individual who hassustained a wound to remove nonviable tissue from a wound site, aprocess referred to as debridement, and also to remove any foreignmatter from the wound site, also referred to as wound cleansing.

Severe wounds, acute wounds, chronic wounds, burns, and ulcers canbenefit from cellular wound dressings. Several artificial skin productsare available for nonhealing wounds or burns such as: Apligraft®(Norvartis), Demagraft®, Biobrane®, Transcyte® (Advance Tissue Science),Integra® Dermal Regeneration Template® (from Integra Life SciencesTechnology), and OrCel®. These products, however, are not designed toaddress the problem of bacterial tissue infiltration and woundspreading.

Unfortunately, systemic antibiotics are not effective for the treatmentof chronic wounds, and are generally not used unless an acute infectionis also present. Current approaches to the treatment of chronic woundsinclude application of topical antibiotics, but such remedies maypromote the advent of antibiotic-resistant bacterial strains and/or maybe ineffective against bacterial biofilms. It therefore may becomeespecially important to use antiseptics when drug resistant bacteria(e.g., methicillin resistant Staphylococcus aureus, or MRSA) aredetected in the wound. There are many antiseptics widely in use, butbacterial populations or subpopulations that are established in somechronic wounds may not respond to these agents, or to any othercurrently available treatments, thus requiring surgical amputation orresection to prevent further spread of the infection within the host,i.e., the undesirable loss of an infected limb or other tissue.Additionally, a number of antiseptics may be toxic to host cells at theconcentrations that may be needed to be effective against an establishedbacterial infection at a chronic wound site, and hence such antisepticsare unsuitable. This problem may be particularly acute in the case ofefforts to clear infections from internal epithelial surfaces, such asrespiratory (e.g., airway, nasopharyngeal and laryngeal paths, tracheal,pulmonary, bronchi, bronchioles, alveoli, etc.) or gastrointestinal(e.g., buccal, esophageal, gastric, intestinal, rectal, anal, etc.)tracts, or other epithelial surfaces.

Particularly problematic are infections composed of bacterial biofilms,a relatively recently recognized organization of bacteria by which free,single-celled (“planktonic”) bacteria assemble by intercellular adhesioninto organized, multi-cellular communities (biofilms) having markedlydifferent patterns of behavior, gene expression, and susceptibility toenvironmental agents including antibiotics. Biofilms may deploybiological defense mechanisms not found in planktonic bacteria, whichmechanisms can protect the biofilm community against antibiotics andhost immune responses. Established biofilms can arrest the wound-healingprocess.

Research into chronic, non-healing wounds has demonstrated thatmicrobial biofilms are readily detectable in a majority of cases, andthe U.S. Centers for Disease Control (CDC) reports that up to 70% ofinfections in the western world are associated with biofilms. It hasbeen reported that biofilms are more common in chronic wounds than acutewounds (James et al., 2008 Wound Rep. and Regen. 16:37-44). Commonmicrobiologic wound contaminants include S. aureus, including MRSA(Methicillin Resistant Staphylococcus aureus), Enterococci, E. coli, P.aeruginosa, Streptococci, and Acinetobacter baumannii. Some of theseorganisms exhibit an ability to survive on non-nutritive clinicalsurfaces for months. S. aureus, has been shown to be viable for fourweeks on dry glass, and for between three and six months on dried bloodand cotton fibers (Domenico et al., 1999 Infect. Immun. 67:664-669).Both E. coli and P. aeruginosa have been shown to survive even longerthan S. aureus on dried blood and cotton fibers (ibid).

Microbial biofilms are associated with substantially increasedresistance to both disinfectants and antibiotics. Biofilm morphologyresults when bacteria and/or fungi attach to surfaces. This attachmenttriggers an altered transcription of genes, resulting in the secretionof a remarkably resilient and difficult to penetrate polysaccharidematrix, protecting the microbes. Biofilms are very resistant to themammalian immune system, in addition to their very substantialresistance to antibiotics. Biofilms are very difficult to eradicate oncethey become established, so preventing biofilm formation is a veryimportant clinical priority. Recent research has shown that open woundscan quickly become contaminated by biofilms. These microbial biofilmsare thought to delay wound healing, and are very likely related to theestablishment of serious wound infections.

The current guidelines for the care for military wounds, for example,specify vigorous and complete irrigation and debridement (BlankenshipCL, Guidelines for care of open combat casualty wounds, Fleet Operationsand Support. U.S. Bureau of Medicine and Surgery). While this earlyintervention is important, it is not adequate to prevent the developmentof infection. Additional therapeutic steps need to be taken followingdebridement to promote healing, reduce the microbial bio-burden, andthereby reduce the chances of establishing wound infections and woundbiofilms.

Because of the complex nature of military traumatic wounds, thepotential for infection is great, particularly considering theintroduction of foreign objects and other environmental contaminatingagents. Both military and clinical environments (including people withinboth of these environments) act as important sources of potentiallypathogenic microbes, particularly to those suffering from open and/orcomplex wounds. Acute and chronic wounds, including surgical andmilitary wounds, have already compromised the body's primary defense andbarrier against infection; the skin. Wounds thus expose the interior ofthe body (a moist and nutritive environment) to opportunistic andpathogenic infections. Many of these infections, particularly persistentwound infections, are likely related to biofilm formation, as has beenshown to be the case with chronic wounds (James et al., 2008). Infectionof wounds in hospitals constitutes one of the most common causes ofnosocomial infection, and wounds acquired in military and naturaldisaster environments are particularly susceptible to microbialcontamination. Military wounds are predisposed to infection because theyare typically associated with tissue damage, tend to be extensive anddeep, may introduce foreign bodies and interfere with local bloodsupply, may be associated with fractures and burns, and may lead toshock and compromised immune defenses.

Skin Architecture and Wound Healing

Maintenance of intact, functioning skin and other epithelial tissues(e.g., generally avascular epithelial surfaces that form barriersbetween an organism and its external environment, such as those found inskin and also found in the linings of respiratory and gastrointestinaltracts, glandular tissues, etc.) is significant to the health andsurvival of humans and other animals. The skin is the largest body organin humans and other higher vertebrates (e.g., mammals), protectingagainst environmental insults through its barrier function, mechanicalstrength and imperviousness to water. As a significant environmentalinterface, skin provides a protective body covering that permitsmaintenance of physiological equilibria.

Skin architecture is well known. Briefly, epidermis, the skin outerlayer, is covered by the stratum corneum, a protective layer of deadepidermal skin cells (e.g., keratinocytes) and extracellular connectivetissue proteins. The epidermis undergoes a continual process of beingsloughed off as it is replaced by new material pushed up from theunderlying epidermal granular cell, spinous cell, and basal cell layers,where continuous cell division and protein synthesis produce new skincells and skin proteins (e.g., keratin, collagen). The dermis liesunderneath the epidermis, and is a site for the elaboration by dermalfibroblasts of connective tissue proteins (e.g., collagen, elastin,etc.) that assemble into extracellular matrix and fibrous structuresthat confer flexibility, strength and elasticity to the skin. Alsopresent in the dermis are nerves, blood vessels, smooth muscle cells,hair follicles and sebaceous glands.

As the body's first line of defense, the skin is a major target forclinical insults such as physical, mechanical, chemical and biological(e.g., xenobiotic, autoimmune) attack that can alter its structure andfunction. The skin is also regarded as an important component ofimmunological defense of the organism. In the skin can be foundmigrating as well as resident white blood cells (e.g., lymphocytes,macrophages, mast cells) and epidermal dendritic (Langerhans) cellshaving potent antigen-presenting activity, which contribute toimmunological protection. Pigmented melanocytes in the basal layerabsorb potentially harmful ultraviolet (UV) radiation. Disruption of theskin presents undesirable risks to a subject, including those associatedwith opportunistic infections, incomplete or inappropriate tissueremodeling, scarring, impaired mobility, pain and/or othercomplications. Like the skin, other epithelial surfaces (e.g.,respiratory tract, gastrointestinal tract and glandular linings) havedefined structural attributes when healthy such that infection or otherdisruptions may present serious health risks.

Damaged or broken skin may result, for example, from wounds such ascuts, scrapes, abrasions, punctures, burns (including chemical burns),infections, temperature extremes, incisions (e.g., surgical incisions),trauma and other injuries. Efficient skin repair via wound healing istherefore clearly desirable in these and similar contexts.

Although skin naturally exhibits remarkable ability for self-repairfollowing many types of damage, there remain a number of contexts inwhich skin healing does not occur rapidly enough and/or in whichinappropriate cellular tissue repair mechanisms result in incompletelyremodeled skin that as a consequence can lack the integrity, barrierproperties, mechanical strength, elasticity, flexibility, or otherdesirable properties of undamaged skin. Skin wound healing thus presentssuch associated challenges, for example, in the context of chronicwounds.

Wound healing occurs in three dynamic and overlapping phases, beginningwith the formation of a fibrin clot. The clot provides a temporaryshield and a reservoir of growth factors that attracts cells into thewound. It also serves as a provisional extracellular matrix (ECM) thatthe cells invade during repair. Intermingled with clot formation is theinflammatory phase, which is characterized by the infiltration ofphagocytes and neutrophils into the wound, which clear the wound ofdebris and bacteria, while releasing growth factors that amplify theearly healing response. The process of restoring the denuded area isinitiated in the proliferation phase of healing and is driven bychemokines, cytokines, and proteases that have been secreted from theimmune cells and are concentrated within the clot. Keratinocytes arestimulated to proliferate and migrate, which forms the new layer ofepithelium that covers the wound while wound angiogenesis deliversoxygen, nutrients, and inflammatory cells to the wounded area. Theremodeling phase is the final phase of wound repair and it is carriedout by the myofibroblasts, which facilitate connective tissuecontraction, increase wound strength, and deposit the ECM that forms thescar (Martin, P. Wound Healing-Aiming for Perfect Skin Regeneration.Science 1997;4:75-80).

Bismuth Thiol—(BT) Based Antiseptics

A number of natural products (e.g., antibiotics) and synthetic chemicalshaving antimicrobial, and in particular antibacterial, properties areknown in the art and have been at least partially characterized bychemical structures and by antimicrobial effects, such as ability tokill microbes (“cidal” effects such as bacteriocidal properties),ability to halt or impair microbial growth (“static” effects such asbacteriostatic properties), or ability to interfere with microbialfunctions such as colonizing or infecting a site, bacterial secretion ofexopolysaccharides and/or conversion from planktonic to biofilmpopulations or expansion of biofilm formation. Antibiotics,disinfectants, antiseptics and the like (including bismuth-thiol or BTcompounds) are discussed, for example, in U.S. Pat. No. 6,582,719,including factors that influence the selection and use of suchcompositions, including, e.g., bacteriocidal or bacteriostaticpotencies, effective concentrations, and risks of toxicity to hosttissues.

Bismuth, a group V metal, is an element that (like silver) possessesantimicrobial properties. Bismuth by itself may not be therapeuticallyuseful and may exhibit certain inappropriate properties, and so mayinstead be typically administered by means of delivery with a complexingagent, carrier, and/or other vehicle, the most common example of whichis Pepto Bismol®, in which bismuth is combined (chelated) withsubsalicylate. Previous research has determined that the combination ofcertain thiol—(—SH, sulfhydryl) containing compounds such as ethanedithiol with bismuth, to provide an exemplary bismuth thiol (BT)compound, improves the antimicrobial potency of bismuth, compared toother bismuth preparations currently available. There are many thiolcompounds that may be used to produce BTs (disclosed, for example, inDomenico et al., 2001 Antimicrob. Agent. Chemotherap. 45(5):1417-1421,Domenico et al., 1997 Antimicrob. Agent. Chemother. 41(8):1697-1703, andin U.S. RE 37,793, U.S. Pat. No. 6,248,371, U.S. Pat. No. 6,086,921, andU.S. Pat. No. 6,380,248; see also, e.g., U.S. Pat. No. 6,582,719) andseveral of these preparations are able to inhibit biofilm formation.

BT compounds have proven activity against MRSA (methicillin resistant S.aureus), MRSE (methicillin resistant S. epidermidis), Mycobacteriumtuberculosis, Mycobacterium avium, drug-resistant P. aeruginosa,enterotoxigenic E. coli, enterohemorrhagic E. coli, Klebsiellapneumoniae, Clostridium difficile, Heliobacter pylori, Legionellapneumophila, Enterococcus faecalis, Enterobacter cloacae, Salmonellatyphimurium, Proteus vulgaris, Yersinia enterocolitica, Vibrio cholerae,and Shigella Flexneri (Domenico et al., 1997 Antimicrob. AgentsChemother. 41:1697-1703). There is also evidence of activity againstcytomegalovirus, herpes simplex virus type 1 (HSV-1) and HSV-2, andyeasts and fungi, such as Candida albicans. BT roles have also beendemonstrated in reducing bacterial pathogenicity, inhibiting or killinga broad spectrum of antibiotic-resistant microbes (gram-positive andgram-negative), preventing biofilm formation, preventing septic shock,treating sepsis, and increasing bacterial susceptibility to antibioticsto which they previously exhibited resistance (see, e.g., Domenico etal., 2001 Agents Chemother. 45:1417-1421; Domenico et al., 2000 Infect.Med. 17:123-127; Domenico et al., 2003 Res. Adv. In Antimicrob. Agents &Chemother. 3:79-85; Domenico et al., 1997 Antimicrob. Agents Chemother.41(8):1697-1703; Domenico et al., 1999 Infect. lmmun. 67:664-669: Huanget al. 1999 J Antimicrob. Chemother. 44:601-605; Veloira et al., 2003 JAntimicrob. Chemother. 52:915-919; Wu et al., 2002 Am J Respir Cell MolBiol. 26:731-738).

Despite the availability of BT compounds for well over a decade,effective selection of appropriate BT compounds for particularinfectious disease indications has remained an elusive goal, wherebehavior of a particular BT against a particular microorganism cannot bepredicted, where synergistic activity of a particular BT and aparticular antibiotic against a particular microorganism cannot bepredicted, where BT effects in vitro may not always predict BT effectsin vivo, and where BT effects against planktonic (single-cell) microbialpopulations may not be predictive of BT effects against microbialcommunities, such as bacteria organized into a biofilm. Additionally,limitations in solubility, tissue permeability, bioavailability,biodistribution and the like may in the cases of some BT compoundshinder the ability to deliver clinical benefit safely and effectively.The presently disclosed invention embodiments address these needs andoffer other related advantages.

BRIEF SUMMARY

As disclosed herein for the first time, and without wishing to be boundby theory, according to certain embodiments described hereinbismuth-thiol (BT) compounds may be used as antiseptic agents for use inthe treatment of acute wounds, chronic wounds, and/or wounds thatcontain bacterial biofilms, and thus may decrease the number of peopleadversely affected by such wounds (e.g., persistent chronic wounds)while also decreasing the cost incurred during treatment of such wounds.Also, in certain embodiments there are contemplated topical formulationsfor treating acute wounds, chronic wounds, and/or wounds or otherepithelial tissue surfaces that contain bacterial biofilms or bacteriarelated to biofilm formation (e.g., bacteria that are capable of formingor otherwise promoting biofilms), which formulations comprise one ormore BT compound and one or more antibiotic compound, as describedherein, where according to non-limiting theory, appropriately selectedcombinations of BT compound(s) and antibiotic(s) based on the presentdisclosure provide heretofore unpredicted synergy in the antibacterial(including anti-biofilm) effects of such formulations, fortherapeutically effective treatment of acute wounds, chronic wounds,and/or wounds that contain bacterial biofilms. Also provided herein forthe first time are unprecedented bismuth-thiol compositions comprisingsubstantially monodisperse microparticulate suspensions, and methods fortheir synthesis and use.

According to certain embodiments of the invention described herein thereis thus provided a bismuth-thiol composition, comprising a plurality ofmicroparticles that comprise a bismuth-thiol (BT) compound,substantially all of said microparticles having a volumetric meandiameter of from about 0.4 μm to about 5 μm, wherein the BT compoundcomprises bismuth or a bismuth salt and a thiol-containing compound. Inanother embodiment there is provided a bismuth-thiol composition,comprising a plurality of microparticles that comprise a bismuth-thiol(BT) compound, substantially all of said microparticles having avolumetric mean diameter of from about 0.4 μm to about 5 μm and beingformed by a process that comprises (a) admixing, under conditions andfor a time sufficient to obtain a solution that is substantially free ofa solid precipitate, (i) an acidic aqueous solution that comprises abismuth salt comprising bismuth at a concentration of at least 50 mM andthat lacks a hydrophilic, polar or organic solubilizer, with (ii)ethanol in an amount sufficient to obtain an admixture that comprises atleast about 5%, 10%, 15%, 20%, 25% or 30% ethanol by volume; and (b)adding to the admixture of (a) an ethanolic solution comprising athiol-containing compound to obtain a reaction solution, wherein thethiol-containing compound is present in the reaction solution at a molarratio of from about 1:3 to about 3:1 relative to the bismuth, underconditions and for a time sufficient for formation of a precipitatewhich comprises the microparticles comprising the BT compound. Incertain embodiments the bismuth salt is Bi(NO₃)₃. In certain embodimentsthe acidic aqueous solution comprises at least 5%, 10%, 15%, 20%, 22% or22.5% bismuth by weight. In certain embodiments the acidic aqueoussolution comprises at least 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%or 5% nitric acid by weight. In certain embodiments the thiol-containingcompound comprises one or more agents selected from 1,2-ethane dithiol,2,3-dimercaptopropanol, pyrithione, dithioerythritol,3,4-dimercaptotoluene, 2,3-butanedithiol, 1,3-propanedithiol,2-hydroxypropane thiol, 1-mercapto-2-propanol, dithioerythritol,alpha-lipoic acid and dithiothreitol.

In another embodiment there is provided a method for preparing abismuth-thiol composition that comprises a plurality of microparticlesthat comprise a bismuth-thiol (BT) compound, substantially all of saidmicroparticles having a volumetric mean diameter of from about 0.4 μm toabout 5 μm, said method comprising the steps of (a) admixing, underconditions and for a time sufficient to obtain a solution that issubstantially free of a solid precipitate, (i) an acidic aqueoussolution that comprises a bismuth salt comprising bismuth at aconcentration of at least 50 mM and that lacks a hydrophilic, polar ororganic solubilizer, with (ii) ethanol in an amount sufficient to obtainan admixture that comprises at least about 5%, 10%, 15%, 20%, 25% or 30%ethanol by volume; and (b) adding to the admixture of (a) an ethanolicsolution comprising a thiol-containing compound to obtain a reactionsolution, wherein the thiol-containing compound is present in thereaction solution at a molar ratio of from about 1:3 to about 3:1relative to the bismuth, under conditions and for a time sufficient forformation of a precipitate which comprises the microparticles comprisingthe BT compound. In certain embodiments the method further comprisesrecovering the precipitate to remove impurities. In certain embodimentsthe bismuth salt is Bi(NO₃)₃. In certain embodiments the acidic aqueoussolution comprises at least 5%, 10%, 15%, 20%, 22% or 22.5% bismuth byweight. In certain embodiments the acidic aqueous solution comprises atleast 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5% nitric acid byweight. In certain embodiments the thiol-containing compound comprisesone or more agents selected from the group consisting of 1,2-ethanedithiol, 2,3-dimercaptopropanol, pyrithione, dithioerythritol, 3,4-dimercaptotoluene, 2,3-butanedithiol, 1,3-propanedithiol,2-hydroxypropane thiol, 1-mercapto-2-propanol, dithioerythritol,dithiothreitol and alpha-lipoic acid.

In another embodiment there is provided a method for protecting anepithelial tissue surface against a bacterial pathogen, comprisingcontacting the epithelial tissue surface with an effective amount of aBT composition under conditions and for a time sufficient for one ormore of (i) prevention of infection of the epithelial tissue surface bythe bacterial pathogen, (ii) inhibition of cell viability or cell growthof substantially all planktonic cells of the bacterial pathogen, (iii)inhibition of biofilm formation by the bacterial pathogen, and (iv)inhibition of biofilm viability or biofilm growth of substantially allbiofilm-form cells of the bacterial pathogen, wherein the BT compositioncomprises a plurality of microparticles that comprise a bismuth-thiol(BT) compound, substantially all of said microparticles having avolumetric mean diameter of from about 0.4 μm to about 5 μm. In certainembodiments the bacterial pathogen is selected from Staphylococcusaureus (S. aureus), MRSA (methicillin-resistant S. aureus),Staphylococcus epidermidis, MRSE (methicillin-resistant S. epidermidis),Mycobacterium tuberculosis, Mycobacterium avium, Pseudomonas aeruginosa,drug-resistant P. aeruginosa, Escherichia coli, enterotoxigenic E. coli,enterohemorrhagic E. coli, Klebsiella pneumoniae, Clostridium difficile,Heliobacter pylori, Legionella pneumophila, Enterococcus faecalis,methicillin-susceptible Enterococcus faecalis, Enterobacter cloacae,Salmonella typhimurium, Proteus vulgaris, Yersinia enterocolitica,Vibrio cholera, Shigella flexneri, vancomycin-resistant Enterococcus(VRE), Burkholderia cepacia complex, Francisella tularensis, Bacillusanthracis, Yersinia pestis, Pseudomonas aeruginosa, vancomycin-resistantenterococci, and Acinetobacter baumannii. In certain embodiments thebacterial pathogen exhibits antibiotic resistance. In certainembodiments the bacterial pathogen exhibits resistance to an antibioticthat is selected from methicillin, vancomycin, naficilin, gentamicin,ampicillin, chloramphenicol, doxycycline and tobramycin.

In certain embodiments the epithelial tissue surface comprises a tissuethat is selected from epidermis, dermis, respiratory tract,gastrointestinal tract and glandular linings. In certain embodiments thestep of contacting is performed one or a plurality of times. In certainembodiments at least one step of contacting comprises one of spraying,irrigating, dipping and painting the epithelial tissue surface. Incertain embodiments at least one step of contacting comprises one ofinhaling, ingesting and orally irrigating. In certain embodiments leastone step of contacting comprises administering by a route that isselected from topically, intraperitoneally, orally, parenterally,intravenously, intraarterially, transdermally, sublingually,subcutaneously, intramuscularly, transbuccally, intranasally, viainhalation, intraoccularly, intraauricularly, intraventricularly,subcutaneously, intraadiposally, intraarticularly and intrathecally. Incertain embodiments the BT composition comprises one or more BTcompounds selected from the group consisting of BisBAL, BisEDT,Bis-dimercaprol, Bis-DTT, Bis-2-mercaptoethanol, Bis-DTE, Bis-Pyr,Bis-Ery, Bis-Tol, Bis-BDT, Bis-PDT, Bis-Pyr/Bal, Bis-Pyr/BDT,Bis-Pyr/EDT, Bis-Pyr/PDT, Bis-Pyr/Tol, Bis-Pyr/Ery,bismuth-1-mercapto-2-propanol, and Bis-EDT/2-hydroxy-1-propanethiol.

In certain embodiments the bacterial pathogen exhibits antibioticresistance. In certain other embodiments the above described methodfurther comprises contacting the epithelial tissue surface with asynergizing antibiotic, simultaneously or sequentially and in any orderwith respect to the step of contacting the epithelial tissue surfacewith the BT composition. In certain embodiments the synergizingantibiotic comprises an antibiotic that is selected from anaminoglycoside antibiotic, a carbapenem antibiotic, a cephalosporinantibiotic, a fluoroquinolone antibiotic, a glycopeptide antibiotic, alincosamide antibiotic, a penicillinase-resistant penicillin antibiotic,and an aminopenicillin antibiotic. In certain embodiments thesynergizing antibiotic is an aminoglycoside antibiotic that is selectedfrom amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin,paromomycin, rhodostreptomycin, streptomycin, tobramycin and apramycin.

In another embodiment of the invention described herein there isprovided a method for overcoming antibiotic resistance (e.g., for abacterial pathogen that is resistant to at least one anti-bacterialeffect of at least one antibiotic known to have an anti-bacterial effectagainst bacteria of the same bacterial species, rendering such apathogen susceptible to an antibiotic) on an epithelial tissue surfacewhere an antibiotic-resistant bacterial pathogen is present, comprisingcontacting the epithelial tissue surface contacting simultaneously orsequentially and in any order with an effective amount of (1) at leastone bismuth-thiol (BT) composition and (2) at least one antibiotic thatis capable of acting synergistically with the at least one BTcomposition, under conditions and for a time sufficient for one or moreof: (i) prevention of infection of the epithelial tissue surface by thebacterial pathogen, (ii) inhibition of cell viability or cell growth ofsubstantially all planktonic cells of the bacterial pathogen, (iii)inhibition of biofilm formation by the bacterial pathogen, and (iv)inhibition of biofilm viability or biofilm growth of substantially allbiofilm-form cells of the bacterial pathogen, wherein the BT compositioncomprises a plurality of microparticles that comprise a bismuth-thiol(BT) compound, substantially all of said microparticles having avolumetric mean diameter of from about 0.4 μm to about 5 μm; and therebyovercoming antibiotic resistance on the epithelial tissue surface. Incertain embodiments the bacterial pathogen is selected fromStaphylococcus aureus (S. aureus), MRSA (methicillin-resistant S.aureus), Staphylococcus epidermidis, MRSE (methicillin-resistant S.epidermidis), Mycobacterium tuberculosis, Mycobacterium avium,Pseudomonas aeruginosa, drug-resistant P. aeruginosa, Escherichia coli,enterotoxigenic E. coli, enterohemorrhagic E. coli, Klebsiellapneumoniae, Clostridium difficile, Heliobacter pylori, Legionellapneumophila, Enterococcus faecalis, methicillin-susceptible Enterococcusfaecalis, Enterobacter cloacae, Salmonella typhimurium, Proteusvulgaris, Yersinia enterocolitica, Vibrio cholera, Shigella flexneri,vancomycin-resistant Enterococcus (VRE), Burkholderia cepacia complex,Francisella tularensis, Bacillus anthracis, Yersinia pestis, Pseudomonasaeruginosa, vancomycin-resistant enterococci, and Acinetobacterbaumannii.

In certain embodiments the bacterial pathogen exhibits resistance to anantibiotic that is selected from methicillin, vancomycin, naficilin,gentamicin, ampicillin, chloramphenicol, doxycycline, tobramycin,clindamicin and gatifloxacin. In certain embodiments the epithelialtissue surface comprises a tissue that is selected from the groupconsisting of epidermis, dermis, respiratory tract, gastrointestinaltract and glandular linings. In certain embodiments the step ofcontacting is performed one or a plurality of times. In certainembodiments at least one step of contacting comprises one of spraying,irrigating, dipping and painting the epithelial tissue surface. Incertain other embodiments at least one step of contacting comprises oneof inhaling, ingesting and orally irrigating. In certain embodiments atleast one step of contacting comprises administering by a route that isselected from topically, intraperitoneally, orally, parenterally,intravenously, intraarterially, transdermally, sublingually,subcutaneously, intramuscularly, transbuccally, intranasally, viainhalation, intraoccularly, intraauricularly, intraventricularly,subcutaneously, intraadiposally, intraarticularly and intrathecally. Incertain embodiments the BT composition comprises one or more BTcompounds selected from BisBAL, BisEDT, Bis-dimercaprol, Bis-DTT,Bis-2-mercaptoethanol, Bis-DTE, Bis-Pyr, Bis-Ery, Bis-Tol, Bis-BDT,Bis-PDT, Bis-Pyr/Bal, Bis-Pyr/BDT, Bis-Pyr/EDT, Bis-Pyr/PDT,Bis-Pyr/Tol, Bis-Pyr/Ery, bismuth-1-mercapto-2-propanol, andBis-EDT/2-hydroxy-1-propanethiol. In certain embodiments the synergizingantibiotic comprises an antibiotic that is selected from clindamicin,gatifloxacin, an aminoglycoside antibiotic, a carbapenem antibiotic, acephalosporin antibiotic, a fluoroquinolone antibiotic, a glycopeptideantibiotic, a lincosamide antibiotic, a penicillinase-resistantpenicillin antibiotic, and an aminopenicillin antibiotic. In certainembodiments the synergizing antibiotic is an aminoglycoside antibioticthat is selected from amikacin, arbekacin, gentamicin, kanamycin,neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin,tobramycin and apramycin.

Turning to another embodiment there is provided a method of treating anacute wound, a chronic wound or a wound or epithelial tissue surfacethat contains bacterial biofilm in a subject, comprising administering,to a wound site or epithelial tissue surface in the subject, atherapeutically effective amount of a topical formulation that comprises(a) at least one BT compound, and (b) a pharmaceutically acceptableexcipient or carrier for topical use. In another embodiment there isprovided a method of treating an acute wound, a chronic wound or a woundor epithelial tissue surface that contains bacterial biofilm in asubject, comprising administering, to a wound site or epithelial tissuesurface in the subject, a therapeutically effective amount of a topicalformulation that comprises (a) at least one BT compound, (b) at leastone antibiotic compound that is capable of acting synergistically withthe BT compound, and (c) a pharmaceutically acceptable excipient orcarrier for topical use.

In certain embodiments the BT compound is selected from BisBAL, BisEDT,Bis-dimercaprol, Bis-DTT, Bis-2-mercaptoethanol, Bis-DTE, Bis-Pyr,Bis-Ery, Bis-Tol, Bis-BDT, Bis-PDT, Bis-Pyr/Bal, Bis-Pyr/BDT,Bis-Pyr/EDT, Bis-Pyr/PDT, Bis-Pyr/Tol, Bis-Pyr/Ery,bismuth-1-mercapto-2-propanol, and Bis-EDT/2-hydroxy-1-propanethiol. Incertain embodiments the BT composition comprises a plurality ofmicroparticles that comprise a bismuth-thiol (BT) compound,substantially all of said microparticles having a volumetric meandiameter of from about 0.4 μm to about 5 μm. In certain embodiments theBT compound is selected from BisEDT and BisBAL. In certain embodimentsthe wound is an acute wound or a chronic wound that contains a bacterialinfection. In certain embodiments the bacterial infection comprises oneor more of gram-positive bacteria and gram-negative bacteria. In certainembodiments the bacterial infection comprises at least one bacterialpopulation selected from a bacterial biofilm and planktonic bacteria. Incertain embodiments the antibiotic compound comprises an antibiotic thatis selected from an aminoglycoside antibiotic, a carbapenem antibiotic,a cephalosporin antibiotic, a fluoroquinolone antibiotic, a glycopeptideantibiotic, a lincosamide antibiotic, a penicillinase-resistantpenicillin antibiotic, and an aminopenicillin antibiotic. In certainembodiments the antibiotic is an aminoglycoside antibiotic that isselected from amikacin, arbekacin, gentamicin, kanamycin, neomycin,netilmicin, paromomycin, rhodostreptomycin, streptomycin, tobramycin andapramycin. In certain embodiments the aminoglycoside antibiotic isamikacin.

Turning to another embodiment there is provided an antisepticcomposition for treating an acute wound, a chronic wound or a wound orepithelial tissue surface that contains bacterial biofilm, comprising(a) at least one BT compound; (b) at least one antibiotic compound thatis capable of acting synergistically with the BT compound; and (c) apharmaceutically acceptable excipient or carrier for topical use. Incertain embodiments the BT compound is selected from BisBAL, BisEDT,Bis-dimercaprol, Bis-DTT, Bis-2-mercaptoethanol, Bis-DTE, Bis-Pyr,Bis-Ery, Bis-Tol, Bis-BDT, Bis-PDT, Bis-Pyr/Bal, Bis-Pyr/BDT,Bis-Pyr/EDT, Bis-Pyr/PDT, Bis-Pyr/Tol, Bis-Pyr/Ery,bismuth-1-mercapto-2-propanol, and Bis-EDT/2-hydroxy-1-propanethiol. Incertain embodiments the BT composition comprises a plurality ofmicroparticles that comprise a bismuth-thiol (BT) compound,substantially all of said microparticles having a volumetric meandiameter of from about 0.4 μm to about 5 μm. In certain embodiments theBT compound is selected from BisEDT and BisBAL. In certain embodimentsthe antibiotic compound comprises an antibiotic that is selected frommethicillin, vancomycin, naficilin, gentamicin, ampicillin,chloramphenicol, doxycycline, tobramycin, clindamicin, gatifloxacin andan aminoglycoside antibiotic. In certain embodiments the aminoglycosideantibiotic is selected from amikacin, arbekacin, gentamicin, kanamycin,neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin,tobramycin and apramycin. In certain embodiments the aminoglycosideantibiotic is amikacin.

In certain other embodiments there is provided a method for treating anacute wound, a chronic wound or a wound or epithelial tissue surfacethat contains bacterial biofilm, comprising (a) identifying a bacterialinfection in a wound or epithelial tissue surface in a subject ascomprising one of (i) gram positive bacteria, (ii) gram negativebacteria, and (iii) both (i) and (ii); (b) administering a topicalformulation that comprises one or more bismuth thiol (BT) compositionsto the wound, wherein (i) if the bacterial infection comprises grampositive bacteria, then the formulation comprises therapeuticallyeffective amounts of at least one BT compound and at least oneantibiotic that is rifamycin, (ii) if the bacterial infection comprisesgram negative bacteria, then the formulation comprises therapeuticallyeffective amounts of at least one BT compound and amikacin, (iii) if thebacterial infection comprises both gram positive and gram negativebacteria, then the formulation comprises therapeutically effectiveamounts of one or a plurality of BT compounds, rifamycin and amikacin,and thereby treating the wound or epithelial tissue surface. In certainembodiments treating the wound prevents neuropathy resulting fromchronic wound progression. In certain embodiments the bacterialinfection comprises one or a plurality of antibiotic-resistant bacteria.In certain embodiments the wound is selected from the group consistingof a venous ulcer, a pressure ulcer, a diabetic ulcer, a decubitisulcer, a gunshot wound, a puncture wound, a shrapnel wound, an ischemicwound, a surgical wound, a traumatic wound, acute arterialinsufficiency, necrotizing fasciitis, osteomyelitis, a wound resultingfrom radiation poisoning, osteoradionecrosis, soft tissue radionecrosis,pyoderma gangrenosum, a gangrenous wound, a burn, a dermal infection anda malignancy. In certain embodiments the wound is an acute wound or achronic wound that comprises a bacterial biofilm. In certain embodimentstreating the wound comprises at least one of: (i) eradicating thebacterial biofilm, (ii) reducing the bacterial biofilm, and (iii)impairing growth of the bacterial biofilm. In certain embodiments the BTcomposition comprises a plurality of microparticles that comprise abismuth-thiol (BT) compound, substantially all of said microparticleshaving a volumetric mean diameter of from about 0.4 μm to about 5 μm.

These and other aspects of the herein described invention embodimentswill be evident upon reference to the following detailed description andattached drawings. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet, includingU.S. RE 37,793, U.S. Pat. No. 6,248,371, U.S. Pat. No. 6,086,921, andU.S. Pat. No. 6,380,248, are incorporated herein by reference in theirentirety, as if each was incorporated individually. Aspects andembodiments of the invention can be modified, if necessary, to employconcepts of the various patents, applications and publications toprovide yet further embodiments.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows surviving numbers (log CFU; colony forming units) fromPseudomonas aeruginosa colony biofilms grown for 24 hours on 10% trypticsoy agar (TSA) at 37° C., followed with indicated treatment for 18hours. Indicated antibiotic treatments are TOB, tobramycin 10X MIC; AMK,amikacin 100X MIC; IPM, imipenem 10X MIC; CEF, cefepime 10X MIC; CIP,ciprofloxacin 100X MIC; Cpd 2B, compound 2B (Bis-BAL, 1:1.5). (MIC;minimum inhibitory concentration, e.g., lowest concentration thatprevents bacterial growth).

FIG. 2 shows surviving numbers (log CFU) from Staphylococcus aureuscolony biofilms grown for 24 hours on 10% tryptic soy agar, followed bythe indicated treatment. Indicated antibiotic treatments are Rifampicin,RIF 100X MIC; daptomycin, DAP 320X MIC; minocycline, MIN 100X MIC;ampicillin, AMC 10X MIC; vancomycin, VAN 10X MIC; Cpd 2B, compound 2B(Bis-BAL, 1:1.5), Cpd 8-2, compound 8-2 (Bis-Pyr/BDT (1:1/0.5).

FIG. 3 shows scratch closure over time of keratinocytes exposed tobiofilms. (*) Significantly different from control (P<0.001).

DETAILED DESCRIPTION

Particular embodiments of the invention disclosed herein are based onthe surprising discovery that certain bismuth-thiol (BT) compounds asprovided herein, but not certain other BT compounds, exhibited potentantiseptic, antibacterial and/or anti-biofilm activity againstparticular bacteria associated with clinically significant infections inacute and/or chronic wounds and/or wounds that contain bacterialbiofilms and/or on epithelial tissue surfaces as provided herein.

Unexpectedly, not all BT compounds were uniformly effective against suchbacteria in a predictable fashion, but instead exhibited differentpotencies depending on the target bacterial species. In particular andas described herein, certain BT compounds were found to exhibit higherpotency against gram-negative bacteria, while certain other BT compoundswere found to exhibit greater potency against gram-positive bacteria, ina manner that, according to non-limiting theory, may for the first timeafford clinically relevant strategies for the management of bacterialinfections, including bacterial biofilm infections, that are present inacute wounds, chronic wounds, and/or other wounds that contain bacterialbiofilms and/or on epithelial tissue surfaces.

Additionally, and as described in greater detail below, certainembodiments of the invention described herein relate to surprisingadvantages that are provided by novel bismuth-thiol (BT) compositionsthat, as disclosed herein, can be made in preparations that comprise aplurality of BT microparticles that are substantially monodisperse withrespect to particle size (e.g., having volumetric mean diameter fromabout 0.4 μm to about 5 μm).

As also disclosed herein, with respect to certain embodiments, it hasbeen discovered that antibacterial and anti-biofilm efficacies ofcertain antibiotics, which antibiotics have previously been found tolack therapeutic effect against such bacterial infections, may besignificantly enhanced (e.g., increased in a statistically significantmanner) by treating the infection (e.g., by direct application on or inan acute or chronic wound site or other epithelial tissue surface) withone or more of these antibiotics in concert with a selected BT compound.In a manner that could not be predicted prior to the present disclosure,certain BT compounds can be combined with certain antibiotics to providea synergizing combination with respect to antibacterial and/oranti-biofilm activity against certain bacterial species or bacterialstrains. The unpredicted nature of such combinations, as described ingreater detail below, is evidenced by the observations that whilecertain BT/antibiotic combinations acted synergistically against certainbacteria, certain other BT/antibiotic combinations failed to exhibitsynergistic antibacterial and/or anti-biofilm activity.

According to these and related embodiments, the antibiotic and the BTcompound may be administered simultaneously or sequentially and ineither order, and it is noteworthy that the specific combinations of oneor more antibiotic and one or more BT compound as disclosed herein fortreatment of a particular infection such as may be found in an acute orchronic wound (e.g., a biofilm formed by gram-negative or gram-positivebacteria) did not exhibit predictable (e.g., merely additive) activitiesbut instead acted in an unexpectedly synergistic fashion, as a functionof the selected antibiotic, the selected BT compound and thespecifically identified target bacteria.

For example, by way of illustration and not limitation, disclosed hereinfor the first time in the context of topical applications such asbacterially infected chronic wounds or other epithelial tissue surfaces,and further in the context of improved substantially monodispersemicroparticulate BT formulations, either or both of a particularantibiotic compound and a particular BT compound may exert limitedantibacterial effects when used alone against a particular bacterialstrain or species, but the combination of both the antibiotic compoundand the BT compound exerts a potent antibacterial effect against thesame bacterial strain or species, which effect is greater in magnitude(with statistical significance) than the simple sum of the effects ofeach compound when used alone, and is therefore believed according tonon-limiting theory to reflect antibiotic-BT synergy. Accordingly, notevery BT compound may synergize with every antibiotic, and not everyantibiotic may synergize with any BT compound, such that antibiotic-BTsynergy generally is not predictable. Instead, and according to certainembodiments as disclosed herein, specific combinations of synergizingantibiotic and BT compounds surprisingly confer potent antibacterialeffects against particular bacteria, including in particularenvironments such as chronic wounds in skin or soft tissues and/orepithelial tissue surfaces, and further including in certain situationsantibacterial effects against biofilms formed by the particularbacteria.

That is, certain BT-synergizing antibiotics are described herein, whichincludes an antibiotic that is capable of acting synergistically with atleast one BT composition that comprises at least one BT compound asprovided herein, where such synergy manifests as a detectable effectthat is greater (i.e., in a statistically significant manner relative toan appropriate control condition) in magnitude than the effect that canbe detected when the antibiotic is present but the BT compound isabsent, and/or when the BT compound is present but the antibiotic isabsent.

Examples of such a detectable effect may in certain embodiments include(i) prevention of infection by a bacterial pathogen, (ii) inhibition ofcell viability or cell growth of substantially all planktonic cells of abacterial pathogen, (iii) inhibition of biofilm formation by a bacterialpathogen, and (iv) inhibition of biofilm viability or biofilm growth ofsubstantially all biofilm-form cells of a bacterial pathogen, but theinvention is not intended to be so limited, such that in othercontemplated embodiments antibiotic-BT synergy may manifest as one ormore detectable effects that may include alteration (e.g., astatistically significant increase or decrease) of one or more otherclinically significant parameters, for example, the degree of resistanceor sensitivity of a bacterial pathogen to one or more antibiotics orother drugs or chemical agents, the degree of resistance or sensitivityof a bacterial pathogen to one or more chemical, physical or mechanicalconditions (e.g., pH, ionic strength, temperature, pressure), and/or thedegree of resistance or sensitivity of a bacterial pathogen to one ormore biological agents (e.g., a virus, another bacterium, a biologicallyactive polynucleotide, an immunocyte or an immunocyte product such as anantibody, cytokine, chemokine, enzyme including degradative enzymes,membrane-disrupting protein, a free radical such as a reactive oxygenspecies, or the like). Persons familiar with the art will appreciatethese and a variety of other criteria by which the effects of particularagents on the structure, function and/or activity of a bacterialpopulation may be determined (e.g., Coico et al. (Eds.), CurrentProtocols in Microbiology, 2008, John Wiley & Sons, Hoboken, N.J.;Schwalbe et al., Antimicrobial Susceptibility Testing Protocols, 2007,CRC Press, Boca Raton, Fla.), for purposes of ascertaining antibiotic-BTsynergy which, as provided herein, is present when the effects of thesynergizing antibiotic-BT combination exceed the mere sum of the effectsobserved when one component of the combination is not present.

For example, in certain embodiments synergy may be determined bydetermining an antibacterial effect such as those described herein usingvarious concentrations of candidate agents (e.g., a BT and an antibioticindividually and in combination) to calculate a fractional inhibitoryconcentration index (FICI) and a fractional bactericidal concentrationindex (FBCI), according to Eliopoulos et al. (Eliopoulos and Moellering,(1996) Antimicrobial combinations. In Antibiotics in Laboratory Medicine(Lorian, V., Ed.), pp. 330-96, Williams and Wilkins, Baltimore, Md.,USA). Synergy may be defined as an FICI or FBCI index of ≤0.5, nointeraction at >0.5-4 and antagonism at >4. (e.g., Odds, FC (2003)Synergy, antagonism, and what the chequerboard puts between them.Journal of Antimicrobial Chemotherapy 52:1). Synergy may also be definedconventionally as ≥4-fold decrease in antibiotic concentration, oralternatively, using fractional inhibitory concentration (FIC) asdescribed, e.g., by Hollander et al. (1998 Antimicrob. Agents Chemother.42:744).

In view of these and related embodiments, there are provided for thefirst time methods for treating acute wounds, chronic wounds, and/orwounds that contain bacterial biofilms, with a therapeutically effectiveamount of a topical formulation that comprises one or more BT compoundsand, optionally, one or more antibiotic compounds. It will beappreciated that based on the present disclosure, certain antibioticsare now contemplated for use in the treatment of acute and/or chronicwounds, where such antibiotics had previously been viewed by personsfamiliar with the art as ineffective against infections of the typefound in acute or chronic wounds, and/or as unsuitable foradministration in a topical formulation such as a topical formulationfor treating an acute or chronic wound.

Certain embodiments thus contemplate compositions that comprise one ormore BT compounds for use as antiseptics. An antiseptic is a substancethat kills or prevents the growth of microorganisms, and may betypically applied to living tissue, distinguishing the class fromdisinfectants, which are usually applied to inanimate objects (Goodmanand Gilman's “The Pharmacological Basis of Therapeutics”, SeventhEdition, Gilman et al., editors, 1985, Macmillan Publishing Co.,(hereafter, Goodman and Gilman”) pp. 959-960). Common examples ofantiseptics are ethyl alcohol and tincture of iodine. Germicides includeantiseptics that kill microbes such as microbial pathogens.

Certain embodiments described herein may contemplate compositions thatcomprise one or more BT compounds and one or more antibiotic compound.Antibiotics are known in the art and typically comprise a drug made froma compound produced by one species of microorganism to kill anotherspecies of microorganism, or a synthetic product having an identical orsimilar chemical structure and mechanism of action, e.g., a drug thatdestroys microorganisms within or on the body of a living organism,including such drug when applied topically. Among embodiments disclosedherein are those in which an antibiotic may belong to one of thefollowing classes: aminoglycosides, carbapenems, cephalosporins,fluoroquinolones, glycopeptide antibiotics, lincosamides (e.g.,clindamycin), penicillinase-resistant penicillins, and aminopenicillins.Compendia of these and other clinically useful antibiotics are availableand known to those familiar with the art (e.g., Washington UniversitySchool of Medicine, The Washington Manual of Medical Therapeutics(32^(nd) Ed.), 2007 Lippincott, Williams and Wilkins, Philadelphia, Pa.;Hauser, AL, Antibiotic Basics for Clinicians, 2007 Lippincott, Williamsand Wilkins, Philadelphia, Pa.).

An exemplary class of antibiotics for use with one or more BT compoundsin certain herein disclosed embodiments is the aminoglycoside class ofantibiotics, which are reviewed in Edson RS, Terrell CL. Theaminoglycosides. Mayo Clin Proc. 1999 May;74(5):519-28. This class ofantibiotics inhibits bacterial growth by impairing bacterial proteinsynthesis, through binding and inactivation of bacterial ribosomalsubunits. In addition to such bacteriostatic properties, aminoglycosidesalso exhibit bacteriocidal effects through disruption of cell walls ingram-negative bacteria.

Aminoglycoside antibiotics include gentamicin, amikacin, streptomycin,and others, and are generally regarded as useful in the treatment ofgram-negative bacteria, mycobacteria and other microbial pathogens,although cases of resistant strains have been reported. Theaminoglycosides are not absorbed through the digestive tract and so arenot generally regarded as being amenable to oral formulations. Amikacin,for example, although often effective against gentamicin-resistantbacterial strains, is typically administered intravenously orintramuscularly, which can cause pain in the patient. Additionally,toxicities associated with aminoglycoside antibiotics such as amikacincan lead to kidney damage and/or irreversible hearing loss.

Despite these properties, certain embodiments disclosed hereincontemplate oral administration of a synergizing BT/antibioticcombination (e.g., where the antibiotic need not be limited to anaminoglycoside) for treatment of an epithelial tissue surface at one ormore locations along the gastrointestinal tract/alimentary canal. Alsocontemplated in certain other embodiments may be use of compositions andmethods described herein as disinfectants, which refers to preparationsthat kill, or block the growth of, microbes on an external surface of aninanimate object.

As also described elsewhere herein, a BT compound may be a compositionthat comprises bismuth or a bismuth salt and a thiol—(e.g., —SH, orsulfhydryl) containing compound, including those that are described(including their methods of preparation) in Domenico et al., 1997Antimicrob. Agent. Chemother. 41(8):1697-1703, Domenico et al., 2001Antimicob.Agent. Chemother. 45(5):1417-1421, and in U.S. RE 37,793, U.S.Pat. No. 6,248,371, U.S. Pat. No. 6,086,921, and U.S. Pat. No.6,380,248; see also, e.g., U.S. Pat. No. 6,582,719. Certain embodimentsare not so limited, however, and may contemplate other BT compounds thatcomprise bismuth or a bismuth salt and a thiol-containing compound. Thethiol-containing compound may contain one, two, three, four, five, sixor more thiol (e.g., —SH) groups. In preferred embodiments the BTcompound comprises bismuth in association with the thiol-containingcompound via ionic bonding and/or as a coordination complex, while insome other embodiments bismuth may be associated with thethiol-containing compound via covalent bonding such as may be found inan organometallic compound. Certain contemplated embodiments, however,expressly exclude a BT compound that is an organometallic compound suchas a compound in which bismuth is found in covalent linkage to anorganic moiety.

Exemplary BT compounds are shown in Table 1:

TABLE 1 Exemplary BT Compounds*  1) CPD 1B-1 Bis-EDT (1:1) BiC₂H₄S₂  2)CPD 1B-2 Bis-EDT (1:1.5) BiC₃H₆S₃  3) CPD 1B-3 Bis-EDT (1:1.5) BiC₃H₆S₃ 4) CPD 1C Bis-EDT (1:1.5) BiC₃H₆S₃  5) CPD 2A Bis-Bal (1:1) BiC₃H₆S₂O 6) CPD 2B Bis-Bal (1:1.5) BiC_(4.5)H₉O_(1.5)S₃  7) CPD 3A Bis-Pyr(1:1.5) BiC_(7.5)H₆N_(1.5)O_(1.5)S_(1.5)  8) CPD 3B Bis-Pyr (1:3)BiC₁₅H₁₂N₃O₃S₃  9) CPD 4 Bis-Ery (1:1.5) BiC₆H₁₂O₃S₃ 10) CPD 5 Bis-Tol(1:1.5) BiC_(10.5)H₉S₃ 11) CPD 6 Bis-BDT (1:1.5) BiC₆H₁₂S₃ 12) CPD 7Bis-PDT (1:1.5) BiC_(4.5)H₉S₃ 13) CPD 8-1 Bis-Pyr/BDT (1:1/1) 14) CPD8-2 Bis-Pyr/BDT (1:1/0.5) 15) CPD 9 Bis-2hydroxy, propane thiol (1:3)16) CPD 10 Bis-Pyr/Bal (1:1/0.5) 17) CPD 11 Bis-Pyr/EDT (1:1/0.5) 18)CPD 12 Bis-Pyr/Tol (1:1/0.5) 19) CPD 13 Bis-Pyr/PDT (1:1/0.5) 20) CPD 14Bis-Pyr/Ery (1:1/0.5) 21) CPD 15 Bis-EDT/2hydroxy, propane thiol (1:1/1)*Shown are atomic ratios relative to a single bismuth atom, forcomparison, based on the stoichiometric ratios of the reactants used andthe known propensity of bismuth to form trivalent complexes with sulfurcontaining compounds. Atomic ratios as shown may not be accuratemolecular formulae for all species in a given preparation. The numbersin parenthesis are the ratios of bismuth to one (or more) thiol agents,(e.g. Bi:thiol1/thiol2) “CPD”, compound.

BT compounds for use in certain of the presently disclosed embodimentsmay be prepared according to established procedures (e.g., U.S. RE37,793, U.S. Pat. No. 6,248,371, U.S. Pat. No. 6,086,921, and U.S. Pat.No. 6,380,248; Domenico et al., 1997 Antimicrob. Agent. Chemother.41(8):1697-1703, Domenico et al., 2001 Antimicob.Agent. Chemother.45(5):1417-1421) and in certain other embodiments BT compounds may alsobe prepared according to methodologies described herein. Certainpreferred embodiments thus contemplate the herein described syntheticmethods for preparing BT compounds, and in particular for obtaining BTcompounds in substantially monodisperse microparticulate form, in whichan acidic aqueous bismuth solution that contains dissolved bismuth at aconcentration of at least 50 mM, at least 100 mM, at least 150 mM, atleast 200 mM, at least 250 mM, at least 300 mM, at least 350 mM, atleast 400 mM, at least 500 mM, at least 600 mM, at least 700 mM, atleast 800 mM, at least 900 mM or at least 1 M and that lacks ahydrophilic, polar or organic solubilizer is admixed with ethanol toobtain a first ethanolic solution, which is reacted with a secondethanolic solution comprising a thiol-containing compound to obtain areaction solution, wherein the thiol-containing compound is present inthe reaction solution at a molar ratio of from about 1:3 to about 3:1relative to the bismuth, under conditions and for a time sufficient forformation of a precipitate which comprises the microparticles comprisingthe BT compound (such as the conditions of concentration, solventstrength, temperature, pH, mixing and/or pressure, and the like, asdescribed herein and as will be appreciated by the skilled person basedon the present disclosure).

Accordingly, exemplary BTs include compound 1B-1, Bis-EDT(bismuth-1,2-ethane dithiol, reactants at 1:1); compound 1B-2, Bis-EDT(1:1.5); compound 1B-3, Bis-EDT (1:1.5); compound 1C, Bis-EDT (solubleBi preparation, 1:1.5); compound 2A, Bis-Bal (bismuth-Britishanti-Lewisite (bismuth-dimercaprol, bismuth-2,3-dimercaptopropanol),1:1); compound 2B, Bis-Bal (1:1.5); compound 3A Bis-Pyr(bismuth-pyrithione, 1:1.5); compound 3B Bis-Pyr (1:3); compound 4,Bis-Ery (bismuth-dithioerythritol, 1:1.5); compound 5, Bis-Tol(bismuth-3,4-dimercaptotoluene, 1:1.5); compound 6, Bis-BDT(bismuth-2,3-butanedithiol, 1:1.5); compound 7, Bis-PDT(bismuth-1,3-propanedithiol, 1:1.5); compound 8-1 Bis-Pyr/BDT (1:1/1);compound 8-2, Bis-Pyr/BDT (1:1/0.5); compound 9, Bis-2-hydroxy, propanethiol (bismuth-1-mercapto-2-propanol, 1:3); compound 10, Bis-Pyr/Bal(1:1/0.5); compound 11, Bis-Pyr/EDT (1:1/0.5); compound 12 Bis-Pyr/Tol(1:1/0.5); compound 13, Bis-Pyr/PDT (1:1/0.5); compound 14 Bis-Pyr/Ery(1:1/0.5); compound 15, Bis-EDT/2-hydroxy, propane thiol (1:1/1) (see,e.g., Table 1).

Without wishing to be bound by theory, it is believed that the presentlydisclosed methods of preparing a BT compound, which in certain preferredembodiments may comprise preparing or obtaining an acidic aqueous liquidsolution that comprises bismuth such as an aqueous nitric acid solutioncomprising bismuth nitrate, may desirably yield compositions comprisingBT compounds where such compositions have one or more desirableproperties, including ease of large-scale production, improved productpurity, uniformity or consistency (including uniformity in particlesize), or other properties useful in the preparation and/oradministration of the present topical formulations.

In particular embodiments it has been discovered that BT compositions,prepared according to the methods described herein for the first time,exhibit an advantageous degree of homogeneity with respect to theiroccurrence as a substantially monodisperse suspension of microparticleseach having a volumetric mean diameter (VMD) according to certainpresently preferred embodiments of from about 0.4 μm to about 5 μm.Measures of particle size can be referred to as volumetric mean diameter(VMD), mass median diameter (MMD), or mass median aerodynamic diameter(MMAD). These measurements may be made, for example, by impaction (MMDand MMAD) or by laser (VMD) characterization. For liquid particles, VMD,MMD and MMAD may be the same if environmental conditions are maintained,e.g., standard humidity. However, if humidity is not maintained, MMD andMMAD determinations will be smaller than VMD due to dehydration duringimpactor measurements. For the purposes of this description, VMD, MMDand MMAD measurements are considered to be under standard conditionssuch that descriptions of VMD, MMD and MMAD will be comparable.Similarly, dry powder particle size determinations in MMD, and MMAD arealso considered comparable.

As described herein, preferred embodiments relate to a substantiallymonodisperse suspension of BT-containing microparticles. Generation of adefined BT particle size with limited geometric standard deviation (GSD)may, for instance, optimize BT deposition, accessibility to desiredtarget sites in an acute wound, a chronic wound or a wound or epithelialtissue surface, and/or tolerability by a subject to whom the BTmicroparticles are administered. Narrow GSD limits the number ofparticles outside the desired VMD or MMAD size range.

In one embodiment, a liquid or aerosol suspension of microparticlescontaining one or more BT compounds disclosed herein is provided havinga VMD from about 0.5 microns to about 5 microns. In another embodiment,a liquid or aerosol suspension having a VMD or MMAD from about 0.7microns to about 4.0 microns is provided. In another embodiment, aliquid or aerosol suspension having aVMD or MMAD from about 1.0 micronto about 3.0 microns is provided. In certain other preferred embodimentsthere is provided a liquid suspension comprising one or a plurality ofBT compound particles of from about 0.1 to about 5.0 microns VMD, or offrom about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6,about 0.7, about 0.8 or about 0.9 microns to about 1.0, about 1.5, about2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0,about 5.5, about 6.0, about 6.5, about 7.0, about 7.5 or about 8.0microns, the particle comprising a BT compound prepared as describedherein.

Accordingly and in certain preferred embodiments, a BT preparationdescribed for the first time herein which is “substantially”monodisperse, for example, a BT composition that comprises a BT compoundin microparticulate form wherein “substantially” all of themicroparticles have a volumetric mean diameter (VMD) within a specifiedrange (e.g., from about 0.4 μm to about 5 μm), includes thosecompositions in which at least 80%, 85%, 90%, 91%, 92%, 93%, or 94%,more preferably at least 95%, 96%, 97%, 98%, 99% or more of theparticles have a VMD that is within the recited size range.

These and related properties of BT compositions prepared according tothe herein described synthetic methods offer unprecedented advantagesover previously described BTs, including lower cost and ease ofproduction, and uniformity within the composition that may permit itscharacterization in a manner that facilitates regulatory complianceaccording to one or more of pharmaceutical, formulary and cosmeceuticalstandards.

Additionally or alternatively, the herein described substantiallymonodisperse BT microparticles may advantageously be produced withoutthe need for micronization, i.e., without the expensive andlabor-intensive milling or supercritical fluid processing or otherequipment and procedures that are typically used to generatemicroparticles (e.g., Martin et al. 2008 Adv. Drug Deliv. Rev.60(3):339; Moribe et al., 2008 Adv. Drug Deliv. Rev. 60(3):328; Cape etal., 2008 Pharm. Res. 25(9):1967; Rasenack et al. 2004 Pharm. Dev.Technol. 9(1):1-13). Hence, the present embodiments offer beneficialeffects of substantially uniform microparticulate preparations,including without limitation enhanced and substantially uniformsolubilization properties, suitability for desired administration formssuch as oral, inhaled or dermatological/skin wound topical forms,increased bioavailability and other beneficial properties.

The BT compound microparticulate suspension can be administered asaqueous formulations, as suspensions or solutions in aqueous as well asorganic solvents including halogenated hydrocarbon propellants, as drypowders, or in other forms as elaborated below, including preparationsthat contain wetting agents, surfactants, mineral oil or otheringredients or additives as may be known to those familiar withformulary, for example, to maintain individual microparticles insuspension. Aqueous formulations may be aerosolized by liquid nebulizersemploying, for instance, either hydraulic or ultrasonic atomization.Propellant-based systems may use suitable pressurized dispensers. Drypowders may use dry powder dispersion devices, which are capable ofdispersing the BT-containing microparticles effectively. A desiredparticle size and distribution may be obtained by choosing anappropriate device.

As also noted above, also provided herein according to certainembodiments is a method for preparing a bismuth-thiol (BT) compositionthat comprises a plurality of microparticles that comprise a BTcompound, substantially all of such microparticles having a volumetricmean diameter (VMD) of from about 0.1 to about 8 microns, and in certainpreferred embodiments from about 0.4 microns to about 5 microns.

In general terms, the method comprises the steps of (a) admixing, underconditions and for a time sufficient to obtain a solution that issubstantially free of a solid precipitate, (i) an acidic aqueoussolution that comprises a bismuth salt comprising bismuth at aconcentration of at least 50 mM and that lacks a hydrophilic, polar ororganic solubilizer, with (ii) ethanol in an amount sufficient to obtainan admixture that comprises at least about 5%, 10%, 15%, 20%, 25% or30%, and preferably about 25% ethanol by volume; and (b) adding to theadmixture of (a) an ethanolic solution comprising a thiol-containingcompound to obtain a reaction solution, wherein the thiol-containingcompound is present in the reaction solution at a molar ratio of fromabout 1:3 to about 3:1 relative to the bismuth, under conditions and fora time sufficient for formation of a precipitate which comprises the BTcompound.

In certain preferred embodiments the bismuth salt may be Bi(NO₃)₃, butit will be appreciated according to the present disclosure that bismuthmay also be provided in other forms. In certain embodiments the bismuthconcentration in the acidic aqueous solution may be at least 100 mM, atleast 150 mM, at least 200 mM, at least 250 mM, at least 300 mM, atleast 350 mM, at least 400 mM, at least 500 mM, at least 600 mM, atleast 700 mM, at least 800 mM, at least 900 mM or at least 1 M. Incertain embodiments the acidic aqueous solution comprises at least 5%,10%, 15%, 20%, 22% or 22.5% bismuth by weight. The acidic aqueoussolution may in certain preferred embodiments comprise at least 5% ormore nitric acid by weight, and in certain other embodiments the acidicaqueous solution may comprise at least 0.5%, at least 1%, at least 1.5%,at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, atleast 4.5% or at least 5% nitric acid by weight.

The thiol-containing compound may be any thiol-containing compound asdescribed herein, and in certain embodiments may comprise one or more of1,2-ethane dithiol, 2,3-dimercaptopropanol, pyrithione,dithioerythritol, 3,4-d imercaptotoluene, 2,3-butanedithiol,1,3-propanedithiol, 2-hydroxypropane thiol, 1-mercapto-2-propanol,dithioerythritol and dithiothreitol. Other exemplary thiol-containingcompounds include alpha-lipoic acid, methanethiol (CH₃SH [m-mercaptan]),ethanethiol (C₂H₅SH [e-mercaptan]), 1-propanethiol (C₃H₇SH [n-Pmercaptan]), 2-Propanethiol (CH₃CH(SH)CH₃ [2C₃ mercaptan]), butanethiol(C₄H₉SH ([n-butyl mercaptan]), tert-butyl mercaptan (C(CH₃)₃SH [t-butylmercaptan]), pentanethiols (C₅H₁₁SH [pentyl mercaptan]), coenzyme A,lipoamide, glutathione, cysteine, cystine, 2-mercaptoethanol,dithiothreitol, dithioerythritol, 2-mercaptoindole, transglutaminase andany of the following thiol compounds available from Sigma-Aldrich (St.Louis, Mo.): (11-mercaptoundecyl)hexa(ethylene glycol),(11-mercaptoundecyl)tetra(ethylene glycol),(11-mercaptoundecyl)tetra(ethylene glycol) functionalized goldnanoparticles, 1,1′,4′,1″-terphenyl-4-thiol, 1,11-undecanedithiol,1,16-hexadecanedithiol, 1,2-ethanedithiol technical grade,1,3-propanedithiol, 1,4-benzenedimethanethiol, 1,4-butanedithiol,1,4-butanedithiol diacetate, 1,5-pentanedithiol, 1,6-hexanedithiol,1,8-octanedithiol, 1,9-nonanedithiol, adamantanethiol, 1-butanethiol,1-decanethiol, 1-dodecanethiol, 1-heptanethiol, 1-heptanethiol purum,1-hexadecanethiol, 1-hexanethiol, 1-mercapto-(triethylene glycol),1-mercapto-(triethylene glycol) methyl ether functionalized goldnanoparticles, 1-mercapto-2-propanol, 1-nonanethiol, 1-octadecanethiol,1-octanethiol, 1-octanethiol, 1-pentadecanethiol, 1-pentanethiol,1-propanethiol, 1-tetradecanethiol, 1-tetradecanethiol purum,1-undecanethiol, 11-(1H-pyrrol-1-yl)undecane-1-thiol,11-amino-1-undecanethiol hydrochloride, 11-bromo-1-undecanethiol,11-mercapto-1-undecanol, 11-mercapto-1-undecanol, 11-mercaptoundecanoicacid, 11-mercaptoundecanoic acid, 11-mercaptoundecyl trifluoroacetate,11-mercaptoundecylphosphoric acid, 12-mercaptododecanoic acid,12-mercaptododecanoic acid, 15-mercaptopentadecanoic acid,16-mercaptohexadecanoic acid, 16-mercaptohexadecanoic acid,1H,1H,2H,2H-perfluorodecanethiol, 2,2′-(ethylenedioxy)diethanethiol,2,3-butanedithiol, 2-butanethiol, 2-ethylhexanethiol,2-methyl-1-propanethiol, 2-methyl-2-propanethiol, 2-phenylethanethiol,3,3,4,4,5,5,6,6,6-nonafluoro-1-hexanethiol purum,3-(dimethoxymethylsilyl)-1-propanethiol, 3-chloro-1-propanethiol,3-mercapto-1-propanol, 3-mercapto-2-butanol,3-mercapto-N-nonylpropionamide, 3-mercaptopropionic acid,3-mercaptopropyl-functionalized silica gel, 3-methyl-1-butanethiol,4,4′-bis(mercaptomethyl)biphenyl, 4,4′-d imercaptostilbene,4-(6-mercaptohexyloxy)benzyl alcohol, 4-cyano-1-butanethiol,4-mercapto-1-butanol, 6-(ferrocenyl)hexanethiol, 6-mercapto-1-hexanol,6-mercaptohexanoic acid, 8-mercapto-1-octanol, 8-mercaptooctanoic acid,9-mercapto-1-nonanol, biphenyl-4,4′-dithiol, butyl 3-mercaptopropionate,copper(I) 1-butanethiolate, cyclohexanethiol, cyclopentanethiol,decanethiol functionalized silver nanoparticles, dodecanethiolfunctionalized gold nanoparticles, dodecanethiol functionalized silvernanoparticles, hexa(ethylene glycol)mono-11-(acetylthio)undecyl ether,mercaptosuccinic acid, methyl 3-mercaptopropionate, nanoTether BPA-HH,NanoThinks™ 18, NanoThinks™ 8, NanoThinks™ ACID11, NanoThinks™ ACID16,NanoThinks™ ALCO11, NanoThinks™ THIO8, octanethiol functionalized goldnanoparticles, PEG dithiol average M_(n) 8,000, PEG dithiol average molwt 1,500, PEG dithiol average mol wt 3,400,S-(11-bromoundecyl)thioacetate, S-(4-cyanobutyl)thioacetate, thiophenol,triethylene glycol mono-11-mercaptoundecyl ether, trimethylolpropanetris(3-mercaptopropionate),[11-(methylcarbonylthio)undecyl]tetra(ethylene glycol),m-carborane-9-thiol, p-terphenyl-4,4″-dithiol, tert-dodecylmercaptan,tert-nonyl mercaptan.

Exemplary reaction conditions, including temperature, pH, reaction time,the use of stirring or agitation to dissolve solutes and procedures forcollecting and washing precipitates, are described herein and employtechniques generally known in the art.

Unlike previously described methodologies for producing BT compounds,according to the present methods for preparing BT, BT products areprovided as microparticulate suspensions having substantially allmicroparticles with VMD from about 0.4 to about 5 microns in certainpreferred embodiments, and generally from about 0.1 microns to about 8microns according to certain other embodiments. Further unlike previousapproaches, according to the instant embodiments bismuth is provided inan acidic aqueous solution that comprises a bismuth salt at aconcentration of from at least about 50 mM to about 1 M, and nitric acidin an amount from at least about 0.5% to about 5% (w/w), and preferablyless than 5% (weight/weight), and that lacks a hydrophilic, polar ororganic solubilizer.

In this regard the present methods offer surprising and unexpectedadvantages in view of generally accepted art teachings that bismuth isnot water soluble at 50 μM (e.g., U.S. RE 37793), that bismuth isunstable in water (e.g., Kuvshinova et al., 2009 Russ. J Inorg. Chem54(11):1816), and that bismuth is unstable even in nitric acid solutionsunless a hydrophilic, polar or organic solubilizer is present. Forexample, in all of the definitive descriptions of BT preparationmethodologies (e.g., Domenico et al., 1997 Antimicrob. Agents.Chemother. 41:1697; U.S. Pat. No. 6,380,248; U.S. RE 37793; U.S. Pat.No. 6,248,371), the hydrophilic solubilizing agent propylene glycol isrequired to dissolve bismuth nitrate, and the bismuth concentration ofsolutions prepared for reaction with thiols is well below 15 mM, therebylimiting the available production modalities for BT compounds.

By contrast, according to the present disclosure there is no requirementfor a hydrophilic, polar or organic solubilizer in order dissolvebismuth, yet higher concentrations are surprisingly achieved.Hydrophilic, polar or organic solubilizers include propylene glycol (PG)and ethylene glycol (EG) and may also include any of a large number ofknown solubility enhancers, including polar solvents such as dioxane anddimethylsulfoxide (DMSO), polyols (including, e.g., PG and EG and alsoincluding polyethylene glycol (PEG), polypropyleneglycol (PPG),pentaerythritol and others), polyhydric alchohols such as glycerol andmannitol, and other agents. Other water-miscible organic of highpolarity include dimethylsulfoxide (DMSO), dimethylformamide (DMF) andNMP (N-methyl-2-pyrrolidone).

Thus, it will be appreciated by those familiar with the art thatsolvents, including those commonly used as hydrophilic, polar or organicsolubilizers as provided herein, may be selected, for instance, based onthe solvent polarity/polarizability (SPP) scale value using the systemof Catalan et al. (e.g., 1995 Liebigs Ann. 241; see also Catalan, 2001In: Handbook of Solvents, Wypych (Ed.), Andrew Publ., NY, and referencescited therein), according to which, for example, water has a SPP valueof 0.962, toluene a SPP value of 0.655, and 2-propanol a SPP value of0.848. Methods for determining the SPP value of a solvent based onultraviolet measurements of the2-N,N-dimethyl-7-nitrofluorene/2-fluoro-7-nitrofluorene probe/homomorphpair have been described (Catalan et al., 1995).

Solvents with desired SPP values (whether as pure single-componentsolvents or as solvent mixtures of two, three, four or more solvents;for solvent miscibility see, e.g., Godfrey 1972 Chem. Technol. 2:359)based on the solubility properties of a particular BT composition can bereadily identified by those having familiarity with the art in view ofthe instant disclosure, although as noted above, according to certainpreferred embodiments regarding the herein described synthetic methodsteps, no hydrophilic, polar or organic solubilizer is required in orderdissolve bismuth.

Solubility parameters may also include the interaction parameter C,Hildebrand solubility parameter d, or partial (Hansen) solubilityparameters: δp, δh and δd, describing the solvent's polarity, hydrogenbonding potential and dispersion force interaction potential,respectively. In certain embodiments, the highest value for a solubilityparameter that describes a solvent or co-solvent system in which thebismuth salt comprising bismuth will dissolve may provide a limitationfor the aqueous solution that comprises the bismuth salt, for instance,according to the presently described method for preparing amicroparticulate BT composition. For example, higher δh values will havea greater hydrogen bonding ability and would therefore have a greateraffinity for solvent molecules such as water. A higher value of maximumobserved δh for a solvent may therefore be preferred for situationswhere a more hydrophilic environment is desired.

By way of non-limiting example, BisEDT having the structure shown belowin formula I may be prepared according to the following reaction scheme:

Briefly, and as a non-limiting illustrative example, to an excess (11.4L) of 5% aqueous HNO₃ at room temperature may be slowly added 0.331 L(about 0.575 moles) of an aqueous acidic bismuth solution such as aBi(NO₃)₃ solution (e.g., 43% Bi(NO₃)₃ (w/w), 5% nitric acid (w/w), 52%water (w/w), available from Shepherd Chemical Co., Cincinnati, Ohio)with stirring, followed by slow addition of absolute ethanol (4 L). Anethanolic solution (1.56

L) of a thiol compound such as 1,2-ethanedithiol [˜0.55 M] may beseparately prepared by adding, to 1.5 L of absolute ethanol, 72.19 mL(0.863 moles) of 1,2-ethanedithiol using a 60 mL syringe, and thenstirring for five minutes. 1,2-ethanedithiol (CAS 540-63-6) and otherthiol compounds are available from, e.g., Sigma-Aldrich, St. Louis, Mo.The ethanolic solution of the thiol compound may then be slowly added tothe aqueous Bi(NO₃)₃/HNO₃ solution with stirring overnight to form areaction solution. The thiol-containing compound may be present in thereaction solution, according to certain preferred embodiments, at amolar ratio of from about 1:3 to about 3:1 relative to the bismuth. Theformed product is allowed to settle as a precipitate comprisingmicroparticles as described herein, which is then collected byfiltration and washed sequentially with ethanol, water and acetone toobtain BisEDT as a yellow amorphous powdered solid. The crude productmay be redissolved in absolute ethanol with stirring, then filtered andwashed sequentially with ethanol several times followed by acetoneseveral times. The washed powder may be triturated in 1M NaOH (500 mL),filtered and washed sequentially with water, ethanol and acetone toafford purified microparticulate BisEDT.

According to non-limiting theory, bismuth inhibits the ability ofbacteria to produce extracellular polymeric substances (EPS) such asbacterial exopolysaccharides, and this inhibition leads to impairedbiofilm formation. Bacteria are believed to employ the glue-like EPS forbiofilm cohesion. Depending on the nature of an infection, biofilmformation and elaboration of EPS may contribute to bacterialpathogenicity such as interference with wound healing. However, bismuthalone is not therapeutically useful as an intervention agent, and isinstead typically administered as part of a complex such as a BT.Bismuth-thiols (BTs) are thus a family of compositions that includescompounds that result from the chelation of bismuth with a thiolcompound, and that exhibit dramatic improvement in the antimicrobialtherapeutic efficacy of bismuth. BTs exhibit remarkable anti-infective,anti-biofilm, and immunomodulatory effects. Bismuth thiols are effectiveagainst a broad-spectrum of microorganisms, and are typically notaffected by antibiotic-resistance. BTs prevent biofilm formation atremarkably low (sub-inhibitory) concentrations, prevent many pathogeniccharacteristics of common wound pathogens at those same sub-inhibitorylevels, can prevent septic shock in animal models, and may besynergistic with many antibiotics.

As described herein, such synergy in the antibacterial effects of one ormore specified BT when combined with one or more specified antibioticcompound is not readily predictable based on profiles of separateantibiotic and BT effects against a particular bacterial type, butsurprisingly may result from selection of particular BT-antibioticcombinations in view of the specific bacterial population, includingidentification of whether gram-negative or gram-positive (or both)bacteria are present. For instance, as disclosed herein, antibioticsthat synergize with certain BTs may include one or more of amikacin,ampicillin, cefazolin, cefepime, chloramphenicol, ciprofloxacin,clindamycin (or other lincosamide antibiotics), daptomycin (Cubicin®),doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin, linezolid(Zyvox®), minocycline, nafcilin, paromomycin, rifampin,sulphamethoxazole, tobramycin and vancomycin. In vitro studies showed,for example, that MRSA, which was poorly or not at all susceptible togentamicin, cefazolin, cefepime, suphamethoxazole, imipenim orlevofloxacin individually, exhibited marked sensitivity to any one ofthese antibiotics if exposed to the antibiotic in the presence of the BTcompound BisEDT. Certain embodiments contemplated herein thus expresslycontemplate compositions and/or methods in which may be included thecombination of a BT compound and one or more antibiotics selected fromamikacin, ampicillin, cefazolin, cefepime, chloramphenicol,ciprofloxacin, clindamycin (or another lincosamide antibiotic),daptomycin (Cubicin®), doxycycline, gatifloxacin, gentamicin, imipenim,levofloxacin, linezolid (Zyvox®), minocycline, nafcilin, paromomycin,rifampin, sulphamethoxazole, tobramycin and vancomycin, whilst certainother embodiments contemplated herein contemplate compositions and/ormethods in which may be included the combination of a BT compound andone or more antibiotics from which expressly excluded may be one or moreantibiotic selected from amikacin, ampicillin, cefazolin, cefepime,chloramphenicol, ciprofloxacin, clindamycin (or other lincosamides),daptomycin (Cubicin®), doxycycline, gatifloxacin, gentamicin, imipenim,levofloxacin, linezolid (Zyvox®), minocycline, nafcilin, paromomycin,rifampin, sulphamethoxazole, tobramycin and vancomycin. It is noted inthis context that gentamicin and tobramycin belong to the aminoglycosideclass of antibiotics. Also expressly excluded from certain contemplatedembodiments are certain compositions and methods described in Domenicoet al., 2001 Agents Chemother. 45:1417-1421; Domenico et al., 2000Infect. Med. 17:123-127; Domenico et al., 2003 Res. Adv. In Antimicrob.Agents & Chemother. 3:79-85; Domenico et al., 1997 Antimicrob. AgentsChemother. 41(8):1697-1703; Domenico et al., 1999 Infect. Immun.67:664-669: Huang et al. 1999 J Antimicrob. Chemother. 44:601-605;Veloira et al., 2003 J Antimicrob. Chemother. 52:915-919; Wu et al.,2002 Am J Respir Cell Mol Biol. 26:731-738; Halwani et al., 2008 Int. JPharm. 358:278).

Accordingly and as described herein, in certain preferred embodimentsthere are provided compositions and methods for promoting healing of anacute wound, a chronic wound, and/or a wound that contains a bacterialbiofilm in a subject, such as skin tissue repair that comprises dermalwound healing. As described herein, persons familiar with the relevantart will recognize appropriate clinical contexts and situations in whichsuch skin tissue repair may be desired, criteria for which areestablished in the medical arts, including inter alia, e.g., surgical,military surgical, dermatological, trauma medicine, gerontological,cardiovascular, metabolic diseases (e.g., diabetes, obesity, etc.),infection and inflammation (including in the epithelial linings of therespiratory tract or the gastrointestinal tract, or other epithelialtissue surfaces such as in glandular tissues), and other relevantmedical specialties and subspecialities. It will therefore beappreciated that, as disclosed herein and known in the art, promotingskin tissue repair (or other epithelial tissue repair) may comprisestimulating or disinhibiting one or more cellular wound repairactivities selected from (i) epithelial cell (e.g., keratinocyte) ordermal fibroblast migration, (ii) epithelial cell (e.g., keratinocyte)or dermal fibroblast growth, (iii) downregulation of epithelial cell(e.g., keratinocyte) or dermal fibroblast collagenase, gelatinase ormatrix metalloproteinase activity, (iv) dermal fibroblast extracellularmatrix protein deposition, and (v) induction or potentiation of dermalangiogenesis. Methodologies for identifying and characterizing suchcellular wound repair activities have been described such that theeffects of the herein disclosed wound tissue repair-promoting compounds,such as compositions comprising BT agents as described herein, on theseand related activities can be determined readily and without undueexperimentation based on the present disclosure. For example, disclosedherein are compositions and methods that relate to art accepted modelsfor wound repair based on keratinocyte wound closure following a scratchwound.

Preferred compositions for treating an acute wound, chronic wound,and/or wound that contains a bacterial biofilm in a subject, to promoteskin tissue repair including wound repair, for use according to theembodiments described herein, may include in certain embodimentscompositions that comprise bismuth-thiol (BT) compounds as describedherein, and which may in certain distinct but related embodiments alsoinclude other compounds that are known in the art such as one or moreantibiotic compounds as described herein. BT compounds and methods formaking them are disclosed herein and are also disclosed, for example, inDomenico et al. (1997 Antimicrob. Agent. Chemother. 41(8):1697-1703;2001 Antimicrob. Agent. Chemother. 45(5)1417-1421) and in U.S. RE37,793, U.S. Pat. No. 6,248,371, U.S. Pat. No. 6,086,921, and U.S. Pat.No. 6,380,248. As also noted above, certain preferred BT compounds arethose that contain bismuth or a bismuth salt ionically bonded to, or ina coordination complex with, a thiol-containing compound, such as acomposition that comprises bismuth chelated to the thiol-containingcompound, and certain other preferred BT compounds are those thatcontain bismuth or a bismuth salt in covalent bond linkage to thethiol-containing compound. Also preferred are substantially monodispersemicroparticulate BT compositions as described herein. Neither fromprevious efforts to promote acute or chronic wound healing includingskin tissue repair, nor from previous characterization in other contextsof any compounds described herein for the first time as having use incompositions and methods for promoting such wound healing, could it bepredicted that the present methods of using such compounds would havewound healing and tissue repair-promoting effects.

According to preferred embodiments there are thus provided methods fortreating an acute wound, a chronic wound, and/or a wound or epithelialtissue surface that contains a bacterial biofilm in a subject,comprising administering to a wound site or epithelial tissue surface inthe subject, a therapeutically effective amount of a topical formulationthat comprises at least one BT compound and a pharmaceuticallyacceptable excipient or carrier for topical use. In certain embodimentsthe method further comprises administering, simultaneously orsequentially and in either order, at least one antibiotic compound. Theantibiotic compound may be an aminoglycoside antibiotic, a carbapenemantibiotic, a cephalosporin antibiotic, a fluoroquinolone antibiotic, aglycopeptides antibiotic, a lincosamide antibiotic, apenicillinase-resistant penicillin antibiotic, or an aminopenicillinantibiotic. Clinically useful antibiotics are described in, e.g.,Washington University School of Medicine, The Washington Manual ofMedical Therapeutics (32^(nd) Ed.), 2007 Lippincott, Williams andWilkins, Philadelphia, Pa.; and in Hauser, Ala., Antibiotic Basics forClinicians, 2007 Lippincott, Williams and Wilkins, Philadelphia, Pa.

As described herein, certain embodiments derive from the unpredictablediscovery that for acute or chronic wounds or other epithelial tissuesurfaces as provided herein (e.g., skin, respiratory tract linings,gastrointestinal tract linings) in which a bacterial infection comprisesgram positive bacteria, a preferred therapeutically effective topicalformulation may comprise a BT compound (e.g., BisEDT,bismuth:1,2-ethanedithiol; BisPyr, bismuth:pyrithione; BisEDT/Pyr,bismuth:1,2-ethanedithiol/pyrithione) and rifamycin, or a BT compoundand daptomycin (Cubicin®, Cubist Pharmaceuticals, Lexington, Ma.), or aBT compound and linezolid (Zyvox®, Pfizer, Inc., NY, N.Y.), or a BTcompound (e.g., BisEDT, bismuth:1,2-ethanedithiol; BisPyr,bismuth:pyrithione; BisEDT/Pyr, bismuth:1,2-ethanedithiol/pyrithione)and one or more of ampicillin, cefazolin, cefepime, chloramphenicol,clindamycin (or another lincosamide antibiotic), daptomycin (Cubicin®),doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin, linezolid(Zyvox®), nafcilin, paromomycin, rifampin, sulphamethoxazole, tobramycinand vancomycin. As also described herein, certain embodiments derivefrom the unpredictable discovery that for acute or chronic wounds inwhich a bacterial infection comprises gram negative bacteria, apreferred therapeutically effective topical formulation may comprise aBT compound and amikacin. Certain related embodiments contemplatetreatment of an acute or chronic wound comprising gram negative bacteriawith a BT compound and another antibiotic, such as anotheraminoglycoside antibiotic, which in certain embodiments is notgentamicin or tobramycin. Accordingly and in view of these embodiments,other related embodiments contemplate identifying one or more bacterialpopulations or subpopulations within a chronic wound site by the wellknown criterion of being gram positive or gram negative, according tomethodologies that are familiar to those skilled in the medicalmicrobiology art, as a step for selecting appropriate antibioticcompound(s) to include in a topical formulation to be administeredaccording to the present methods.

The presently described compositions and methods may find use in thetreatment of acute and chronic wounds and wound biofilms, including, forexample, as burn creams, as topicals for the treatment of existingwounds including those described herein, for prevention of chronicwounds, for treatment of MRSA skin infections, and for other relatedindications as disclosed herein and as will be apparent to the skilledperson in view of the present disclosure.

Non-limiting examples of bacteria against which the herein describedcompositions and methods may find beneficial use, according to certainembodiments as described herein, include Staphylococcus aureus (S.aureus), MRSA (methicillin-resistant S. aureus), Staphylococcusepidermidis, MRSE (methicillin-resistant S. epidermidis), Mycobacteriumtuberculosis, Mycobacterium avium, Pseudomonas aeruginosa,drug-resistant P. aeruginosa, Escherichia coli, enterotoxigenic E. coli,enterohemorrhagic E. coli, Klebsiella pneumoniae, Clostridium difficile,Heliobacter pylori, Legionella pneumophila, Enterococcus faecalis,methicillin-susceptible Enterococcus faecalis, Enterobacter cloacae,Salmonella typhimurium, Proteus vulgaris, Yersinia enterocolitica,Vibrio cholera, Shigella flexneri, vancomycin-resistant Enterococcus(VRE), Burkholderia cepacia complex, Francisella tularensis, Bacillusanthracis, Yersinia pestis, Pseudomonas aeruginosa, vancomycin-sensitiveand vancomycin-resistant enterococci (e.g., E. faecalis, E. faecium),methicillin-sensitive and methicillin-resistant staphylococci (e.g., S.aureus, S. epidermidis) and Acinetobacter baumannii, Staphylococcushaemolyticus, Staphylococcus hominis, Enterococcus faecium,Streptococcus pyogenes, Streptococcus agalactiae, Bacillus anthracis,Klebsiella pneumonia, Proteus mirabilis, Proteus vulgaris, Yersiniaenterocolytica, Stenotrophomonas maltophilia, and E. cloacae.

The practice of certain embodiments of the present invention willemploy, unless indicated specifically to the contrary, conventionalmethods of microbiology, molecular biology, biochemistry, cell biology,virology and immunology techniques that are within the skill of the art,and reference to several of which is made below for the purpose ofillustration. Such techniques are explained fully in the literature.See, e.g., Sambrook, et al. Molecular Cloning: A Laboratory Manual (2ndEdition, 1989); Maniatis et al. Molecular Cloning: A Laboratory Manual(1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic AcidHybridization (B. Hames & S. Higgins, eds., 1985); Transcription andTranslation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R.Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning(1984).

Unless the context requires otherwise, throughout the presentspecification and claims, the word “comprise” and variations thereof,such as, “comprises” and “comprising” are to be construed in an open,inclusive sense, that is as “including, but not limited to”.

Reference throughout this specification to “one embodiment” or “anembodiment” or “an aspect” means that a particular feature, structure orcharacteristic described in connection with the embodiment is includedin at least one embodiment of the present invention. Thus, theappearances of the phrases “in one embodiment” or “in an embodiment” invarious places throughout this specification are not necessarily allreferring to the same embodiment. Furthermore, the particular features,structures, or characteristics may be combined in any suitable manner inone or more embodiments.

Certain embodiments relate to methods, compositions and kits fortreating an acute or chronic wound or a wound biofilm in a subject,which may comprise promoting skin tissue repair in the subject, or foraltering one or more cellular wound repair activity in a cell orplurality of cells. A cell generally indicates a single cell, whereas aplurality of cells indicates more than one cell. The cells may comprisea tissue, organ or entire organism. Furthermore, the cell or cells maybe located in vivo, in vitro, or ex vivo. Maintaining cell, tissue andorgan cultures are routine procedures for one of skill in the art, theconditions and media for which can be easily ascertained. (See, forexample, Freshney, Culture of Animal Cells: A Manual of Basic Technique,Wiley-Liss 5^(th) Ed.(2005); Davis, Basic Cell Culture, OxfordUniversity Press 2^(nd) Ed. (2002)).

As disclosed herein, certain embodiments relate to methods for treatingan acute or chronic wound or a wound biofilm in a subject that comprisesadministering to the subject a therapeutically effective amount of acomposition comprising a BT compound as described herein for use in suchmethod (e.g., as provided in the form of a plurality of substantiallymonodisperse microparticles), and optionally in certain furtherembodiments also comprising an antibiotic compound as described hereinfor use in such method, for example, a BT compound such as BisEDT orBisBAL or other compounds presented in Table 1 herein, or any other BTagent such as those described in Domenico et al. (1997 Antimicrob.Agent. Chemother. 41:1697; 2001 Antimicrob. Agent. Chemother. 45:1421)and/or in U.S. RE 37,793, U.S. Pat. No. 6,248,371, U.S. Pat. No.6,086,921, and U.S. Pat. No. 6,380,248 and/or as prepared according tothe methods disclosed herein. The step of administering may be performedby any means known to the art, for example, topically (including viadirect administration to skin or to any epithelial tissue surface,including such surfaces as may be present in glandular tissues or in therespiratory and/or gastrointestinal tracts), vaginally,intraperitoneally, orally, parenterally, intravenously, intraarterially,transdermally, sublingually, subcutaneously, intramuscularly,transbuccally, intranasally, via inhalation, intraoccularly,subcutaneously, intraadiposally, intraarticularly or intrathecally.

In preferred embodiments administering may be performed topically, wherepharmaceutical excipients or carriers for topical use are describedherein and known in the art.

As noted above, certain invention embodiments described herein relate totopical formulations of the described BT compounds (e.g., BisEDT and/orBisBAL), which formulations may in certain further embodiments compriseone or more antibiotic compounds as described herein, for instance,amikacin, ampicillin, cefazolin, cefepime, chloramphenicol,ciprofloxacin, clindamycin (or another lincosamide antibiotic),daptomycin (Cubicin®), doxycycline, gatifloxacin, gentamicin, imipenim,levofloxacin, linezolid (Zyvox®), minocycline, nafcilin, paromomycin,rifampin, sulphamethoxazole, tobramycin and vancomycin; or a carbapenemantibiotic, a cephalosporin antibiotic, a fluoroquinolone antibiotic, aglycopeptide antibiotic, a lincosamide antibiotic, apenicillinase-resistant penicillin antibiotic, and/or an aminopenicillinantibiotic, and/or an aminoglycoside antibiotic such as amikacin,arbekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin,rhodostreptomycin, streptomycin, tobramycin or apramycin, and/or alipopeptide antibiotic such as daptomycin (Cubicin®), or anoxazolidinone antibiotic such as linezolid (Zyvox®). These and relatedformulations may comprise the BT compound(s) (and optionally one or moreantibiotics) in a pharmaceutically acceptable carrier, excipient ordiluent and in a therapeutic amount, as disclosed herein, whenadministered topically to an animal, preferably a mammal, and mostpreferably a human, and in particularly preferred embodiments, a humanhaving an acute or chronic wound or a wound that contains a bacterialinfection which may be biofilm-related (e.g., in which bacteria capableof promoting biofilm formation may be present but a biofilm is not yetdetectable) or that contains a bacterial infection such as a biofilm orother bacterial presence.

Topical administration of the BT compounds described herein, or theirpharmaceutically acceptable salts, in pure form or in an appropriatepharmaceutical composition, can be carried out via any of the acceptedmodes of topical administration of agents for serving similar utilities.Topical application or administration of a composition includes, inpreferred embodiments, directly contacting the composition (e.g., atopical formulation) with skin and/or another epithelial tissue surface(e.g., respiratory tract, gastrointestinal tract and/or glandularepithelial linings) of the subject undergoing treatment, which may be atone or more localized or widely distributed skin and/or other epithelialtissue surface sites and which may generally refer to contacting thetopical formulation with an acute or chronic wound site that issurrounded by intact stratum corneum or epidermis but need not be solimited; for instance, certain embodiments contemplate as a topicalapplication the administration of a topical formulation described hereinto injured, abraded or damaged skin, or skin of a subject undergoingsurgery, such that contact of the topical formulation may take place notonly with stratum corneum or epidermis but also with skin granular cell,spinous cell, and/or basal cell layers, and/or with dermal or underlyingtissues, for example, as may accompany certain types of wound repair orwound healing or other skin tissue remodeling.

Such skin tissue repair may therefore comprise, in certain preferredembodiments, dermal wound healing, as may be desirable, for example, inpreventing or ameliorating an acute chronic wound or a wound biofilm or,as another example, in preventing or ameliorating skin wound dehiscence,or in improving, accelerating or otherwise enhancing dermal woundhealing when an acute or chronic wound and/or skin wound dehiscence maybe present. Certain other embodiments that contemplate topicaladministration to an epithelial tissue surface present in respiratorytract, gastrointestinal tract and/or glandular linings similarly maycomprise administration of the topical formulation by an appropriateroute as will be known in the art for delivering a topical preparationas provided herein, to one or more epithelial tissue surfaces present inrespiratory (e.g., airway, nasopharyngeal and laryngeal paths, tracheal,pulmonary, bronchi, bronchioles, alveoli, etc.) and/or gastrointestinal(e.g., buccal, esophageal, gastric, intestinal, rectal, anal, etc.)tracts, and/or other epithelial surfaces.

According to certain contemplated embodiments topical administration maycomprise direct application into an open wound. For instance, an openfracture or other open wound may include a break in the skin that mayexpose additional underlying tissues to the external environment in amanner that renders them susceptible to microbial infection. Such asituation is not uncommon in certain types of acute traumatic militarywounds, including, for example, Type III (severe) open fractures. Inaccord with these and related embodiments, topical administration may beby direct contact of the herein described BT composition with suchdamaged skin and/or another epithelial surface and/or with othertissues, such as, for instance, connective tissues including muscle,ligaments, tendons, bones, circulatory tissues such as blood vessels,associated nerve tissues, and any other organs that may be exposed insuch open wounds. Examples of other tissues that may be exposed, andhence for which such direct contact is contemplated, include kidney,bladder, liver, pancreas, and any other tissue or organ that may be sodetrimentally exposed to opportunistic infection in relation to an openwound.

The topical formulations (e.g., pharmaceutical compositions) may beprepared by combining the described BT compound (e.g., comprising acompound described in U.S. RE 37,793, U.S. Pat. No. 6,248,371, U.S. Pat.No. 6,086,921, and/or U.S. Pat. No. 6,380,248 and/or prepared accordingto the present disclosure such as the herein described microparticulateBT suspensions), and in certain related embodiments by combining one ormore desired antibiotics (e.g., an aminoglycoside antibiotic such asamikacin) separately or together with the BT compound, with anappropriate pharmaceutically acceptable carrier, diluent or excipientfor use in a topical formulation preparation, and may be formulated intopreparations in solid, semi-solid, gel, cream, colloid, suspension orliquid or other topically applied forms, such as powders, granules,ointments, solutions, washes, gels, pastes, plasters, paints,bioadhesives, microsphere suspensions, and aerosol sprays.

Pharmaceutical compositions of these and related embodiments areformulated so as to allow the active ingredients contained therein, andin particularly preferred embodiments the herein described BTcompound(s) alone or in combination with one or more desired antibiotics(e.g., a carbapenem antibiotic, a cephalosporin antibiotic, afluoroquinolone antibiotic, a glycopeptide antibiotic, a lincosamideantibiotic, a penicillinase-resistant penicillin antibiotic, and anaminopenicillin antibiotic, or an aminoglycoside antibiotic such asamikacin, or rifamycin) which may be applied simultaneously orsequentially and in either order, to be bioavailable upon topicaladministration of the formulation containing the BT compound(s) and/orantibiotic composition(s) to an acute or chronic wound and optionally tosurrounding skin of a subject, such as a mammal, including a human, andin certain preferred embodiments a human patient having an acute orchronic wound, or being at increased risk for having, an acute orchronic wound or a wound biofilm or wound dehiscence (e.g., an obeseand/or diabetic individual). Certain embodiments disclosed hereincontemplate topical administration of a BT compound and of anantibiotic, including administration that may be simultaneous orsequential and in either order, but the invention is not intended to beso limited and in other embodiments expressly contemplates a distinctroute of administration for the BT compound relative to the route ofadministration of the antibiotic. Thus, the antibiotic may beadministered orally, intravenously, or by any other route ofadministration as described herein, while the BT compound may beadministered by a route that is independent of the route used for theantibiotic. As a non-limiting, illustrative example, the BT compound maybe administered topically as provided herein, while the antibiotic maybe simultaneously or sequentially (and in any order) administered by adistinct route, such as orally, intravenously, transdermally,subcutaneously, intramuscularly and/or by any other route ofadministration.

The topical formulations described herein deliver a therapeuticallyeffective amount of the antiseptic or wound-healing agent(s) (andoptionally the antibiotic(s)) to the wound site, for instance, to skincells such as dermal fibroblasts. Preferred formulations may becontacted with a desired site such as a topical wound site, a chronicwound, an epithelial tissue surface or other intended site ofadministration by spraying, irrigating, dipping and/or painting; suchformulations therefore may exhibit ready permeability into the skin, ascan be determined according to any of a number of establishedmethodologies known to the art for testing the skin permeability of adrug composition (see, e.g., Wagner et al., 2002 J. Invest. Dermatol.118:540, and references cited therein; Bronaugh et al., 1985 J. Pharm.Sci. 74:64; Bosman et al., 1998 J. Pharm. Biomed. Anal. 17:493-499;Bosman et al., 1996 J. Pharm Biomed Anal. 1996 14:1015-23; Bonferoni etal., 1999 Pharm. Dev. Technol. 4:45-53; Frantz, Instrumentation andmethodology for in vitro skin diffusion cells in methodology for skinabsorption. In: Methods for Skin Absorption (Kemppainen & Reifenrath,Eds), CRC Press, Florida, 1990, pp. 35-59; Tojo, Design and calibrationof in vitro permeation apparatus. In: Transdermal Controlled SystemicMedications (Chien YW, Ed), Marcel Dekker, New York, 1987, 127-158;Barry, Methods for studying percutaneous absorption. In: DermatologicalFormulations: Percutaneous absorption, Marcel Dekker, New York, 1983,234-295).

Compositions, and formulations comprising such compositions, that willbe administered to the skin of a subject or patient may in certainembodiments take the form of one or more dosage units, where forexample, a liquid-filled capsule or ampule may contain a single dosageunit, and a container of a topical formulation as described herein inaerosol form may hold a plurality of dosage units. Actual methods ofpreparing such dosage forms are known, or will be apparent, to thoseskilled in this art; for example, see The Science and Practice ofPharmacy, 20th Edition (Philadelphia College of Pharmacy and Science,2000). The composition or formulation to be administered will, in anyevent, contain a therapeutically effective amount of an antisepticand/or wound healing-promoting compound as provided herein (e.g., a BTcompound), or a pharmaceutically acceptable salt thereof, in accordancewith the present teachings.

As noted above, the present topical formulations may take any of a widevariety of forms, and include, for example, creams, lotions, solutions,sprays, gels, ointments, pastes or the like, and/or may be prepared soas to contain liposomes, micelles, and/or microspheres. See, e.g., U.S.Pat. No. 7,205,003. For instance, creams, as is well known in the artsof pharmaceutical and cosmeceutical formulation, are viscous liquids orsemisolid emulsions, either oil-in-water or water-in-oil. Cream basesare water-washable, and contain an oil phase, an emulsifier, and anaqueous phase. The oil phase, also called the “internal” phase, isgenerally comprised of petrolatum and a fatty alcohol such as cetyl orstearyl alcohol. The aqueous phase usually, although not necessarily,exceeds the oil phase in volume, and generally contains a humectant. Theemulsifier in a cream formulation is generally a nonionic, anionic,cationic or amphoteric surfactant.

Lotions, which are preferred for delivery of cosmetic agents, arepreparations to be applied to the skin surface without friction, and aretypically liquid or semi-liquid preparations in which solid particles,including the active agent, are present in a water or alcohol base.Lotions are usually suspensions of solids, and preferably comprise aliquid oily emulsion of the oil-in-water type. Lotions are preferredformulations herein for treating large body areas, because of the easeof applying a more fluid composition. It is generally preferred that theinsoluble matter in a lotion be finely divided. Lotions will typicallycontain suspending agents to produce better dispersions as well ascompounds useful for localizing and holding the active agent in contactwith the skin, e.g., methylcellulose, sodium carboxymethyl-cellulose, orthe like.

Solutions are homogeneous mixtures prepared by dissolving one or morechemical substances (solutes) in a liquid such that the molecules of thedissolved substance are dispersed among those of the solvent. Thesolution may contain other pharmaceutically acceptable and/orcosmeceutically acceptable chemicals to buffer, stabilize or preservethe solute. Common examples of solvents used in preparing solutions areethanol, water, propylene glycol or any other pharmaceuticallyacceptable and/or cosmeceutically acceptable vehicles.

Gels are semisolid, suspension-type systems. Single-phase gels containorganic macromolecules distributed substantially uniformly throughoutthe carrier liquid, which is typically aqueous, but also, preferably,contain an alcohol, and, optionally, an oil. Preferred “organicmacromolecules,” i.e., gelling agents, may be chemically crosslinkedpolymers such as crosslinked acrylic acid polymers, for instance, the“carbomer” family of polymers, e.g., carboxypolyalkylenes, that may beobtained commercially under the Carbopol® trademark. Also preferred incertain embodiments may be hydrophilic polymers such as polyethyleneoxides, polyoxyethylene-polyoxypropylene copolymers andpolyvinylalcohol; cellulosic polymers such as hydroxypropyl cellulose,hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropylmethylcellulose phthalate, and methyl cellulose; gums such as tragacanthand xanthan gum; sodium alginate; and gelatin. In order to prepare auniform gel, dispersing agents such as alcohol or glycerin can be added,or the gelling agent can be dispersed by trituration, mechanical mixingor stirring, or combinations thereof.

Ointments, as also well known in the art, are semisolid preparationsthat are typically based on petrolatum or other petroleum derivatives.The specific ointment base to be used, as will be appreciated by thoseskilled in the art, is one that will provide for a number of desirablecharacteristics, e.g., emolliency or the like. As with other carriers orvehicles, an ointment base should be inert, stable, nonirritating, andnonsensitizing. As explained in Remington: The Science and Practice ofPharmacy, 19th Ed. (Easton, Pa.: Mack Publishing Co., 1995), at pages1399-1404, ointment bases may be grouped in four classes: oleaginousbases; emulsifiable bases; emulsion bases; and water-soluble bases.Oleaginous ointment bases include, for example, vegetable oils, fatsobtained from animals, and semisolid hydrocarbons obtained frompetroleum. Emulsifiable ointment bases, also known as absorbent ointmentbases, contain little or no water and include, for example,hydroxystearin sulfate, anhydrous lanolin, and hydrophilic petrolatum.Emulsion ointment bases are either water-in-oil (W/O) emulsions oroil-in-water (O/W) emulsions, and include, for example, cetyl alcohol,glyceryl monostearate, lanolin, and stearic acid. Preferredwater-soluble ointment bases are prepared from polyethylene glycols ofvarying molecular weight (see, e.g., Remington, Id.).

Pastes are semisolid dosage forms in which the active agent is suspendedin a suitable base. Depending on the nature of the base, pastes aredivided between fatty pastes or those made from single-phase aqueousgels. The base in a fatty paste is generally petrolatum or hydrophilicpetrolatum or the like. The pastes made from single-phase aqueous gelsgenerally incorporate carboxymethylcellulose or the like as a base.

Formulations may also be prepared with liposomes, micelles, andmicrospheres. Liposomes are microscopic vesicles having one(unilamellar) or a plurality (multilamellar) of lipid walls comprising alipid bilayer, and, in the present context, may encapsulate and/or haveadsorbed to their lipid membranous surfaces one or more components ofthe topical formulations herein described, such as the antiseptic, woundhealing/skin tissue/epithelial tissue repair-promoting compounds (e.g.,microparticulate BT compounds, optionally along with one or moreantibiotics) or certain carriers or excipients. Liposomal preparationsherein include cationic (positively charged), anionic (negativelycharged), and neutral preparations. Cationic liposomes are readilyavailable. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areavailable under the tradename Lipofectin® (GIBCO BRL, Grand Island,N.Y.). Similarly, anionic and neutral liposomes are readily available aswell, e.g., from Avanti Polar Lipids (Birmingham, Ala.), or can beeasily prepared using readily available materials. Such materialsinclude phosphatidyl choline, cholesterol, phosphatidyl ethanolamine,dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol(DOPG), and dioleoylphoshatidyl ethanolamine (DOPE), among others. Thesematerials can also be mixed with DOTMA in appropriate ratios. Methodsfor making liposomes using these materials are well known in the art.

Micelles are known in the art as comprised of surfactant moleculesarranged so that their polar headgroups form an outer spherical shell,while the hydrophobic, hydrocarbon chains are oriented towards thecenter of the sphere, forming a core. Micelles form in an aqueoussolution containing surfactant at a high enough concentration so thatmicelles naturally result. Surfactants useful for forming micellesinclude, but are not limited to, potassium laurate, sodium octanesulfonate, sodium decane sulfonate, sodium dodecane sulfonate, sodiumlauryl sulfate, docusate sodium, decyltrimethylammonium bromide,dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide,tetradecyltrimethyl-ammonium chloride, dodecylammonium chloride,polyoxyl-8 dodecyl ether, polyoxyl-12 dodecyl ether, nonoxynol 10, andnonoxynol 30.

Microspheres, similarly, may be incorporated into the presentlydescribed topical formulations. Like liposomes and micelles,microspheres essentially encapsulate one or more components of thepresent formulations. They are generally, but not necessarily, formedfrom lipids, preferably charged lipids such as phospholipids.Preparation of lipidic microspheres is well known in the art.

Various additives, as known to those skilled in the art, may also beincluded in the topical formulations. For example, solvents, includingrelatively small amounts of alcohol, may be used to solubilize certainformulation components. It may be desirable, for certain topicalformulations or in cases of particularly severe skin injury such as apost-surgical acute or chronic wound or post-surgical dermal wounddehiscence, to include in the topical formulation an added skinpermeation enhancer in the formulation. Examples of suitable enhancersinclude, but are not limited to, ethers such as diethylene glycolmonoethyl ether (available commercially as Transcutol®) and diethyleneglycol monomethyl ether; surfactants such as sodium laurate, sodiumlauryl sulfate, cetyltrimethylammonium bromide, benzalkonium chloride,Poloxamer® (231, 182, 184), Tween® (20, 40, 60, 80), and lecithin (U.S.Pat. No. 4,783,450); alcohols such as ethanol, propanol, octanol, benzylalcohol, and the like; polyethylene glycol and esters thereof such aspolyethylene glycol monolaurate (PEGML; see, e.g., U.S. Pat. No.4,568,343); amides and other nitrogenous compounds such as urea,dimethylacetamide (DMA), dimethylformamide (DMF), 2-pyrrolidone,1-methyl-2-pyrrolidone, ethanolamine, diethanolamine, andtriethanolamine; terpenes; alkanones; and organic acids, particularlycitric acid and succinic acid. Azone® and sulfoxides such as DMSO andC₁₀MSO may also be used, but are less preferred.

Most preferred skin permeation enhancers are those lipophilicco-enhancers typically referred to as “plasticizing” enhancers, i.e.,enhancers that have a molecular weight in the range of about 150 to 1000daltons, an aqueous solubility of less than about 1 wt %, preferablyless than about 0.5 wt %, and most preferably less than about 0.2 wt %.The Hildebrand solubility parameter of plasticizing enhancers is in therange of about 2.5 to about 10, preferably in the range of about 5 toabout 10. Preferred lipophilic enhancers are fatty esters, fattyalcohols, and fatty ethers. Examples of specific and most preferredfatty acid esters include methyl laurate, ethyl oleate, propylene glycolmonolaurate, propylene glycerol dilaurate, glycerol monolaurate,glycerol monooleate, isopropyl n-decanoate, and octyldodecyl myristate.Fatty alcohols include, for example, stearyl alcohol and oleyl alcohol,while fatty ethers include compounds wherein a diol or triol, preferablya C₂-C₄ alkane diol or triol, are substituted with one or two fattyether substituents. Additional skin permeation enhancers will be knownto those of ordinary skill in the art of topical drug delivery, and/orare described in the relevant literature. See, e.g., PercutaneousPenetration Enhancers, eds. Smith et al. (CRC Press, Boca Raton, Fla.,1995).

Various other additives may be included in the topical formulationsaccording to certain embodiments of the present invention, in additionto those identified above. These include, but are not limited to,antioxidants, astringents, perfumes, preservatives, emollients,pigments, dyes, humectants, propellants, and sunscreen agents, as wellas other classes of materials whose presence may be cosmetically,medicinally or otherwise desirable. Typical examples of optionaladditives for inclusion in the formulations of certain embodiments ofthe invention are as follows: preservatives such as sorbate; solventssuch as isopropanol and propylene glycol; astringents such as mentholand ethanol; emollients such as polyalkylene methyl glucosides;humectants such as glycerine; emulsifiers such as glycerol stearate,PEG-100 stearate, polyglyceryl-3 hydroxylauryl ether, and polysorbate60; sorbitol and other polyhydroxyalcohols such as polyethylene glycol;sunscreen agents such as octyl methoxyl cinnamate (availablecommercially as Parsol MCX) and butyl methoxy benzoylmethane (availableunder the tradename Parsol 1789); antioxidants such as ascorbic acid(vitamin C), α-tocopherol (Vitamin E), β-tocopherol, γ-tocopherol,δ-tocopherol, ε-tocopherol, ζ₁-tocopherol, ζ₂-tocopherol, η-tocopherol,and retinol (vitamin A); essential oils, ceramides, essential fattyacids, mineral oils, wetting agents and other surfactants such as thePLURONIC® series of hydrophilic polymers available from BASF (Mt. Olive,N.J.), vegetable oils (e.g., soy bean oil, palm oil, liquid fraction ofshea butter, sunflower oil), animal oils (e.g., perhydrosqualene),mineral oils, synthetic oils, silicone oils or waxes (e.g.,cyclomethicone and dimethicone), fluorinated oils (generallyperfluoropolyethers), fatty alcohols (e.g., cetyl alcohol), and waxes(e.g., beeswax, carnauba wax, and paraffin wax); skin-feel modifiers;and thickeners and structurants such as swelling clays and cross-linkedcarboxypolyalkylenes that may be obtained commercially under theCarbopol® trademark.

Other additives include beneficial agents such as those materials thatcondition the skin (particularly, the upper layers of the skin in thestratum corneum) and keep it soft by retarding the decrease of its watercontent and/or protect the skin. Such conditioners and moisturizingagents include, by way of example, pyrrolidine carboxylic acid and aminoacids; organic antimicrobial agents such as 2,4,4′-trichloro-2-hydroxydiphenyl ether (triclosan) and benzoic acid; anti-inflammatory agentssuch as acetylsalicylic acid and glycyrrhetinic acid; anti-seborrhoeicagents such as retinoic acid; vasodilators such as nicotinic acid;inhibitors of melanogenesis such as kojic acid; and mixtures thereof.Other advantageously included cosmeceutically active agents may bepresent, for example, α-hydroxyacids, α-ketoacids, polymerichydroxyacids, moisturizers, collagen, marine extracts, and antioxidantssuch as ascorbic acid (vitamin C), α-tocopherol (Vitamin E) or othertocopherols such as those described above, and retinol (vitamin A),and/or cosmetically acceptable salts, esters, amides, or otherderivatives thereof. Additional cosmetic agents include those that arecapable of improving oxygen supply in skin tissue, as described, forexample, in WO 94/00098 and WO 94/00109. Sunscreens may also beincluded.

Other embodiments may include a variety of non-carcinogenic,non-irritating healing materials that facilitate treatment with theformulations of certain embodiments of the invention. Such healingmaterials may include nutrients, minerals, vitamins, electrolytes,enzymes, herbs, plant extracts, honey, glandular or animal extracts, orsafe therapeutic agents that may be added to the formulation tofacilitate dermal healing. The amounts of these various additives arethose conventionally used in the cosmetics field, and range, forexample, from about 0.01% to about 20% of the total weight of thetopical formulation.

The formulations of certain embodiments of the invention may alsoinclude conventional additives such as opacifiers, fragrance, colorant,gelling agents, thickening agents, stabilizers, surfactants, and thelike. Other agents may also be added, such as antimicrobial agents, toprevent spoilage upon storage, i.e., to inhibit growth of microbes suchas yeasts and molds. Suitable antimicrobial agents are typicallyselected from methyl and propyl esters of p-hydroxybenzoic acid (e.g.,methyl and propyl paraben), sodium benzoate, sorbic acid, imidurea, andcombinations thereof. The formulations may also containirritation-mitigating additives to minimize or eliminate the possibilityof skin irritation or skin damage resulting from the anti-infectiveacute or chronic wound healing and skin tissue repair-promoting compoundto be administered, or from other components of the composition.Suitable irritation-mitigating additives include, for example:α-tocopherol; monoamine oxidase inhibitors, particularly phenyl alcoholssuch as 2-phenyl-1-ethanol; glycerin; salicylates; ascorbates;ionophores such as monensin; amphiphilic amines; ammonium chloride;N-acetylcysteine; capsaicin; and chioroquine. The irritation-mitigatingadditive, if present, may be incorporated into the topical formulationat a concentration effective to mitigate irritation or skin damage,typically representing not more than about 20 wt %, more typically notmore than about 5 wt %, of the formulation.

The topical formulations may also contain, in addition to theantiseptic/wound healing/anti-biofilm/skin tissue repair-promotingcompound (e.g., a BT compound, preferably as substantially homogeneousmicroparticles as provided herein, and optionally in combination withone or more synergizing antibiotics as described herein), atherapeutically effective amount of one or more additionalpharmacologically active agents suitable for topical administration.Such agents may include an asymmetrical lamellar aggregate consisting ofphospholipids and oxygen-loaded fluorocarbon or a fluorocarbon compoundmixture, which are capable of improving oxygen supply in skin tissue, asdescribed, for example, in International Patent Publication Nos. WO94/00098 and WO 94/00109.

Suitable pharmacologically active agents that may be incorporated intothe present topical formulations and thus topically applied, may includebut are not limited to, the following: agents that improve or eradicatepigmented or non-pigmented age spots, keratoses, and wrinkles;antimicrobial agents; antibacterial agents; antipruritic and antixeroticagents; antiinflammatory agents; local anesthetics and analgesics;corticosteroids; retinoids (e.g., retinoic acid; vitamins; hormones; andantimetabolites. Some examples of topical pharmacologically activeagents include acyclovir, amphotericins, chlorhexidine, clotrimazole,ketoconazole, econazole, miconazole, metronidazole, minocycline,nystatin, neomycin, kanamycin, phenytoin, para-amino benzoic acidesters, octyl methoxycinnamate, octyl salicylate, oxybenzone,dioxybenzone, tocopherol, tocopheryl acetate, selenium sulfide, zincpyrithione, diphenhydramine, pramoxine, lidocaine, procaine,erythromycin, tetracycline, clindamycin, crotamiton, hydroquinone andits monomethyl and benzyl ethers, naproxen, ibuprofen, cromolyn,retinoic acid, retinol, retinyl palmitate, retinyl acetate, coal tar,griseofulvin, estradiol, hydrocortisone, hydrocortisone 21-acetate,hydrocortisone 17-valerate, hydrocortisone 17-butyrate, progesterone,betamethasone valerate, betamethasone dipropionate, triamcinoloneacetonide, fluocinonide, clobetasol propionate, minoxidil, dipyridamole,diphenylhydantoin, benzoyl peroxide, and 5-fluorouracil. As also notedabove, certain embodiments contemplate inclusion in the formulation ofan antibiotic such as a carbapenem antibiotic, a cephalosporinantibiotic, a fluoroquinolone antibiotic, a glycopeptide antibiotic, alincosamide antibiotic, a penicillinase-resistant penicillin antibiotic,an aminopenicillin antibiotic, or an aminoglycoside antibiotic such asamikacin.

A pharmacologically acceptable carrier may also be incorporated in thetopical formulation of certain present embodiments and may be anycarrier conventionally used in the art. Examples include water, loweralcohols, higher alcohols, honey, polyhydric alcohols, monosaccharides,disaccharides, polysaccharides, sugar alcohols such as, for example,glycols (2-carbon), glycerols (3-carbon), erythritols and threitols(4-carbon), arabitols, xylitols and ribitols (5-carbon), mannitols,sorbitols, dulcitols and iditols (6-carbon), isomaltols, maltitols,lactitols and polyglycitols, hydrocarbon oils, fats and oils, waxes,fatty acids, silicone oils, nonionic surfactants, ionic surfactants,silicone surfactants, and water-based mixtures and emulsion-basedmixtures of such carriers.

Topical formulation embodiments of the present invention may be appliedregularly to whatever acute or chronic wound site (e.g., the wounditself and surrounding tissue, including surrounding tissue that appearsunaffected by infection or otherwise normal or healthy) or skin area orother epithelial tissue surface (e.g., gastrointestinal tract,respiratory tract, glandular tissue) requires treatment with thefrequency and in the amount necessary to achieve the desired results.The frequency of treatment depends on the nature of the skin (or otherepithelial tissue) condition (e.g., an acute or chronic wound or otherskin wound such as may be found in dehiscence that results from asurgical incision, or other types of skin wounds), the degree of damageor deterioration of the skin (or other tissue), the responsiveness ofthe user's skin (or other tissue), the strength of the activeingredients (e.g., the herein describedwound-healing/antiseptic/anti-biofilm/skin tissue repair-promotingcompounds such as a BT compound and optionally one or more additionalpharmaceutically active ingredients, such as an antibiotic, e.g.,amikacin or other antibiotic) in the particular embodiment, theeffectiveness of the vehicle used to deliver the active ingredients intothe appropriate layer of the skin (or other epithelialsurface-containing tissue), the ease with which the formula is removedby physical contact with bandages or other dressings or clothing, or itsremoval by sweat or other intrinsic or extrinsic fluids, and theconvenience to the subject's or patient's activity level or lifestyle.

Typical concentrations of active substances such as the BT compoundantiseptic/anti-biofilm/wound-healing/skin tissue repair-promotingcompositions described herein can range, for example, from about0.001-30% by weight based on the total weight of the composition, toabout 0.01-5.0%, and more preferably to about 0.1-2.0%. As onerepresentative example, compositions of these embodiments of the presentinvention may be applied to an acute or chronic wound and/or to the skinat a rate equal to from about 1.0 mg/cm² of skin to about 20.0 mg/cm² ofskin. Representative examples of topical formulations include, but arenot limited to, aerosols, alcohols, anhydrous bases (such as lipsticksand powders), aqeuous solutions, creams, emulsions (including eitherwater-in-oil or oil-in-water emulsions), fats, foams, gels,hydro-alcoholic solutions, liposomes, lotions, microemulsions,ointments, oils, organic solvents, polyols, polymers, powders, salts,silicone derivatives, and waxes. Topical formulations may include, forexample, chelating agents, conditioning agents, emollients, excipients,humectants, protective agents, thickening agents, or UV absorbingagents. One skilled in the art will appreciate that formulations otherthan those listed may be used in embodiments of the present invention.

Chelating agents may be optionally included in topical formulations, andmay be selected from any agent that is suitable for use in a cosmeticcomposition, and may include any natural or synthetic chemical which hasthe ability to bind divalent cationic metals such as Ca²⁺, Mn²⁺, orMg²⁺. Examples of chelating agents include, but are not limited to EDTA,disodium EDTA, EGTA, citric acid, and dicarboxylic acids.

Conditioning agents may also be optionally included in topicalformulations. Examples of skin conditioning agents include, but are notlimited to, acetyl cysteine, N-acetyl dihydrosphingosine,acrylates/behenyl acrylate/dimethicone acrylate copolymer, adenosine,adenosine cyclic phosphate, adensosine phosphate, adenosinetriphosphate, alanine, albumen, algae extract, allantoin andderiviatives, aloe barbadensis extracts, aluminum PCA, amyloglucosidase,arbutin, arginine, azulene, bromelain, buttermilk powder, butyleneglycol, caffeine, calcium gluconate, capsaicin, carbocysteine,carnosine, beta-carotene, casein, catalase, cephalins, ceramides,chamomilla recutita (matricaria) flower extract, cholecalciferol,cholesteryl esters, coco-betaine, coenzyme A, corn starch modified,crystallins, cycloethoxymethicone, cysteine DNA, cytochrome C,darutoside, dextran sulfate, dimethicone copolyols, dimethylsilanolhyaluronate, DNA, elastin, elastin amino acids, epidermal growth factor,ergocalciferol, ergosterol, ethylhexyl PCA, fibronectin, folic acid,gelatin, gliadin, beta-glucan, glucose, glycine, glycogen, glycolipids,glycoproteins, glycosaminoglycans, glycosphingolipids, horseradishperoxidase, hydrogenated proteins, hydrolyzed proteins, jojoba oil,keratin, keratin amino acids, and kinetin, lactoferrin, lanosterol,lauryl PCA, lecithin, linoleic acid, linolenic acid, lipase, lysine,lysozyme, malt extract, maltodextrin, melanin, methionine, mineralsalts, niacin, niacinamide, oat amino acids, oryzanol, palmitoylhydrolyzed proteins, pancreatin, papain, PEG, pepsin, phospholipids,phytosterols, placental enzymes, placental lipids, pyridoxal5-phosphate, quercetin, resorcinol acetate, riboflavin, RNA,saccharomyces lysate extract, silk amino acids, sphingolipids,stearamidopropyl betaine, stearyl palmitate, tocopherol, tocopherylacetate, tocopheryl linoleate, ubiquinone, vitis vinifera (grape) seedoil, wheat amino acids, xanthan gum, and zinc gluconate. Skinconditioning agents other than those listed above may be combined with adisclosed composition or preparation provided thereby, as can be readilyappreciated by one skilled in the art.

Topical formulations may also optionally include one or more emollients,examples of which include, but are not limited to, acetylated lanolin,acetylated lanolin alcohol, acrylates/C₁₀₋₃₀ alkyl acrylatecrosspolymer, acrylates copolymer, alanine, algae extract, aloebarbadensis extract or gel, althea officinalis extract, aluminum starchoctenylsuccinate, aluminum stearate, apricot (prunus armeniaca) kerneloil, arginine, arginine aspartate, arnica montana extract, ascorbicacid, ascorbyl palmitate, aspartic acid, avocado (persea gratissima)oil, barium sulfate, barrier sphingolipids, butyl alcohol, beeswax,behenyl alcohol, beta-sitosterol, BHT, birch (betula alba) bark extract,borage (borago officinalis) extract, 2-bromo-2-nitropropane-1,3-diol,butcherbroom (ruscus aculeatus) extract, butylene glycol, calendulaofficinalis extract, calendula officinalis oil, candelilla (euphorbiacerifera) wax, canola oil, caprylic/capric triglyceride, cardamon(elettaria cardamomum) oil, carnauba (copernicia cerifera) wax,carrageenan (chondrus crispus), carrot (daucus carota sativa) oil,castor (ricinus communis) oil, ceramides, ceresin, ceteareth-5,ceteareth-12, ceteareth-20, cetearyl octanoate, ceteth-20, ceteth-24,cetyl acetate, cetyl octanoate, cetyl palmitate, chamomile (anthemisnobilis) oil, cholesterol, cholesterol esters, cholesterylhydroxystearate, citric acid, clary (salvia sclarea) oil, cocoa(theobroma cacao) butter, coco-caprylate/caprate, coconut (cocosnucifera) oil, collagen, collagen amino acids, corn (zea mays) oil,fatty acids, decyl oleate, dextrin, diazolidinyl urea, dimethiconecopolyol, dimethiconol, dioctyl adipate, dioctyl succinate,dipentaerythrityl hexacaprylate/hexacaprate, DMDM hydantoin, DNA,erythritol, ethoxydiglycol, ethyl linoleate, eucalyptus globulus oil,evening primrose (oenothera biennis) oil, fatty acids, tructose,gelatin, geranium maculatum oil, glucosamine, glucose glutamate,glutamic acid, glycereth-26, glycerin, glycerol, glyceryl distearate,glyceryl hydroxystearate, glyceryl laurate, glyceryl linoleate, glycerylmyristate, glyceryl oleate, glyceryl stearate, glyceryl stearate SE,glycine, glycol stearate, glycol stearate SE, glycosaminoglycans, grape(vitis vinifera) seed oil, hazel (corylus americana) nut oil, hazel(corylus avellana) nut oil, hexylene glycol, honey, hyaluronic acid,hybrid safflower (carthamus tinctorius) oil, hydrogenated castor oil,hydrogenated coco-glycerides, hydrogenated coconut oil, hydrogenatedlanolin, hydrogenated lecithin, hydrogenated palm glyceride,hydrogenated palm kernel oil, hydrogenated soybean oil, hydrogenatedtallow glyceride, hydrogenated vegetable oil, hydrolyzed collagen,hydrolyzed elastin, hydrolyzed glycosaminoglycans, hydrolyzed keratin,hydrolyzed soy protein, hydroxylated lanolin, hydroxyproline,imidazolidinyl urea, iodopropynyl butylcarbamate, isocetyl stearate,isocetyl stearoyl stearate, isodecyl oleate, isopropyl isostearate,isopropyl lanolate, isopropyl myristate, isopropyl palmitate, isopropylstearate, isostearamide DEA, isostearic acid, isostearyl lactate,isostearyl neopentanoate, jasmine (jasminum officinale) oil, jojoba(buxus chinensis) oil, kelp, kukui (aleurites moluccana) nut oil,lactamide MEA, laneth-16, laneth-10 acetate, lanolin, lanolin acid,lanolin alcohol, lanolin oil, lanolin wax, lavender (lavandulaangustifolia) oil, lecithin, lemon (citrus medica limonum) oil, linoleicacid, linolenic acid, macadamia ternifolia nut oil, magnesium stearate,magnesium sulfate, maltitol, matricaria (chamomilla recutita) oil,methyl glucose sesquistearate, methylsilanol PCA, microcrystalline wax,mineral oil, mink oil, mortierella oil, myristyl lactate, myristylmyristate, myristyl propionate, neopentyl glycol dicaprylate/dicaprate,octyldodecanol, octyldodecyl myristate, octyldodecyl stearoyl stearate,octyl hydroxystearate, octyl palmitate, octyl salicylate, octylstearate, oleic acid, olive (olea europaea) oil, orange (citrusaurantium dulcis) oil, palm (elaeis guineensis) oil, palmitic acid,pantethine, panthenol, panthenyl ethyl ether, paraffin, PCA, peach(prunus persica) kernel oil, peanut (arachis hypogaea) oil, PEG-8 C12 18ester, PEG-15 cocamine, PEG-150 distearate, PEG-60 glyceryl isostearate,PEG-5 glyceryl stearate, PEG-30 glyceryl stearate, PEG-7 hydrogenatedcastor oil, PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castoroil, PEG-20 methyl glucose sesquistearate, PEG-40 sorbitan peroleate,PEG-5 soy sterol, PEG-10 soy sterol, PEG-2 stearate, PEG-8 stearate,PEG-20 stearate, PEG-32 stearate, PEG-40 stearate, PEG-50 stearate,PEG-100 stearate, PEG-150 stearate, pentadecalactone, peppermint (menthapiperita) oil, petrolatum, phospholipids, polyamino sugar condensate,polyglyceryl-3 diisostearate, polyquaternium-24, polysorbate 20,polysorbate 40, polysorbate 60, polysorbate 80, polysorbate 85,potassium myristate, potassium palmitate, potassium sorbate, potassiumstearate, propylene glycol, propylene glycol dicaprylate/dicaprate,propylene glycol dioctanoate, propylene glycol dipelargonate, propyleneglycol laurate, propylene glycol stearate, propylene glycol stearate SE,PVP, pyridoxine dipalmitate, quaternium-15, quaternium-18 hectorite,quaternium-22, retinol, retinyl palmitate, rice (oryza sativa) bran oil,RNA, rosemary (rosmarinus officinalis) oil, rose oil, safflower(carthamus tinctorius) oil, sage (salvia officinalis) oil, salicylicacid, sandalwood (santalum album) oil, serine, serum protein, sesame(sesamum indicum) oil, shea butter (butyrospermum parkii), silk powder,sodium chondroitin sulfate, sodium DNA, sodium hyaluronate, sodiumlactate, sodium palmitate, sodium PCA, sodium polyglutamate, sodiumstearate, soluble collagen, sorbic acid, sorbitan laurate, sorbitanoleate, sorbitan palmitate, sorbitan sesquioleate, sorbitan stearate,sorbitol, soybean (glycine soja) oil, sphingolipids, squalane, squalene,stearamide MEA-stearate, stearic acid, stearoxy dimethicone,stearoxytrimethylsilane, stearyl alcohol, stearyl glycyrrhetinate,stearyl heptanoate, stearyl stearate, sunflower (helianthus annuus) seedoil, sweet almond (prunus amygdalus dulcis) oil, synthetic beeswax,tocopherol, tocopheryl acetate, tocopheryl linoleate, tribehenin,tridecyl neopentanoate, tridecyl stearate, triethanolamine, tristearin,urea, vegetable oil, water, waxes, wheat (triticum vulgare) germ oil,and ylang ylang (cananga odorata) oil.

In some embodiments a topical formulation may contain a suitableexcipient, which typically should have a high affinity for the skin, bewell tolerated, stable, and yield a consistency that allows for easyutilization. Suitable topical excipients and vehicles can be routinelyselected for a particular use by those skilled in the art, andespecially with reference to one of many standard texts in the art, suchas Remington's Pharmaceutical Sciences, Vol. 18, Mack Publishing Co.,Easton, Pa. (1990), in particular Chapter 87. Optionally one or morehumectants are also included in the topical formulation. Examples ofhumectants include, but are not limited to, amino acids, chondroitinsulfate, diglycerin, erythritol, fructose, glucose, glycerin, glycerol,glycol, 1,2,6-hexanetriol, honey, hyaluronic acid, hydrogenated honey,hydrogenated starch hydrolysate, inositol, lactitol, maltitol, maltose,mannitol, natural moisturization factor, PEG-15 butanediol, polyglycerylsorbitol, salts of pyrollidone carboxylic acid, potassium PCA, propyleneglycol, sodium glucuronate, sodium PCA, sorbitol, sucrose, trehalose,urea, and xylitol.

Certain embodiments contemplate topical formulations containing one ormore additional skin protective agent. Examples of skin protectiveagents may include, but are not limited to, algae extract, allantoin,aluminum hydroxide, aluminum sulfate, betaine, camellia sinensis leafextract, cerebrosides, dimethicone, glucuronolactone, glycerin, kaolin,lanolin, malt extract, mineral oil, petrolatum, potassium gluconate, andtalc. One skilled in the art will readily appreciate that skinprotectants other than those listed above may also be combined with adisclosed composition of the present invention or preparation providedthereby.

Surfactants may also desirably be included in certain topicalformulations contemplated herein, and can be selected from any naturalor synthetic surfactants suitable for use in cosmetic compositions, suchas cationic, anionic, zwitterionic, or non-ionic surfactants, ormixtures thereof. (See Rosen, M., “Surfactants and InterfacialPhenomena,” Second Edition, John Wiley & Sons, New York, 1988, Chapter1, pages 4 31). Examples of cationic surfactants may include, but arenot limited to, DMDAO or other amine oxides, long-chain primary amines,diamines and polyamines and their salts, quaternary ammonium salts,polyoxyethylenated long-chain amines, and quaternized polyoxyethylenatedlong-chain amines. Examples of anionic surfactants may include, but arenot limited to, SDS; salts of carboxylic acids (e.g., soaps); salts ofsulfonic acids, salts of sulfuric acid, phosphoric and polyphosphoricacid esters; alkylphosphates; monoalkyl phosphate (MAP); and salts ofperfluorocarboxylic acids. Examples of zwitterionic surfactants mayinclude, but are not limited to, cocoamidopropyl hydroxysultaine (CAPHS)and others which are pH-sensitive and require special care in designingthe appropriate pH of the formula (La, alkylaminopropionic acids,imidazoline carboxylates, and betaines) or those which are notpH-sensitive (e.g., sulfobetaines, sultaines). Examples of non-ionicsurfactants may include, but are not limited to, alkylphenolethoxylates, alcohol ethoxylates, polyoxyethylenated polyoxypropyleneglycols, polyoxyethylenated mercaptans, long-chain carboxylic acidesters, alkonolamides, tertiary acetylenic glycols, polyoxyethylenatedsilicones, N-alkylpyrrolidones, and alkylpolyglycosidases. Wettingagents, mineral oil or other surfactants such as non-ionic detergents oragents such as one or more members of the PLURONICS® series (BASF, Mt.Olive, N.J.) may also be included, for example and according tonon-limiting theory, to discourage aggregation of BT microparticleswithin the microparticulate suspension. Any combination of surfactantsis acceptable. Certain embodiments may include at least one anionic andone cationic surfactant, or at least one cationic and one zwitterionicsurfactant which are compatible, i.e., do not form complexes whichprecipitate appreciably when mixed.

Examples of thickening agents that may also be present in certaintopical formulations include, but are not limited to, acrylamidescopolymer, agarose, amylopectin, bentonite, calcium alginate, calciumcarboxymethyl cellulose, carbomer, carboxymethyl chitin, cellulose gum,dextrin, gelatin, hydrogenated tallow, hydroxytheylcellulose,hydroxypropylcellulose, hydroxpropyl starch, magnesium alginate,methylcellulose, microcrystalline cellulose, pectin, various PEG's,polyacrylic acid, polymethacrylic acid, polyvinyl alcohol, variousPPG's, sodium acrylates copolymer, sodium carrageenan, xanthan gum, andyeast beta-glucan. Thickening agents other than those listed above mayalso be used in embodiments of this invention.

According to certain embodiments contemplated herein, a topicalformulation may comprise one or more sunscreening or UV absorbingagents. Where ultraviolet light—(UVA and UVB) absorbing properties aredesired, such agents may include, for example, benzophenone,benzophenone-1, benzophenone-2, benzophenone-3, benzophenone-4,benzophenone-5, benzophenone-6, benzophenone-7, benzophenone-8,benzophenone-9, benzophenone-10, benzophenone-11, benzophenone-12,benzyl salicylate, butyl PABA, cinnamate esters, cinoxate,DEA-methoxycinnamate, diisopropyl methyl cinnamate, ethyldihydroxypropyl PABA, ethyl diisopropylcinnamate, ethylmethoxycinnamate, ethyl PABA, ethyl urocanate, glyceryl octanoatedimethoxycinnamate, glyceryl PABA, glycol salicylate, homosalate,isoamyl p-methoxycinnamate, oxides of titanium, zinc, zirconium,silicon, manganese, and cerium, PABA, PABA esters, Parsol 1789, andisopropylbenzyl salicylate, and mixtures thereof. One skilled in the artwill appreciate that sunscreening and UV absorbing or protective agentsother than those listed may be used in certain embodiments of thepresent invention.

Topical formulations disclosed herein are typically effective at pHvalues between about 2.5 and about 10.0. Preferably, the pH of thecomposition is at or about the following pH ranges: about pH 5.5 toabout pH 8.5, about pH 5 to about pH 10, about pH 5 to about pH 9, aboutpH 5 to about pH 8, about pH 3 to about pH 10, about pH 3 to about pH 9,about pH 3 to about pH 8, and about pH 3 to about pH 8.5. Mostpreferably, the pH is about pH 7 to about pH 8. One of ordinary skill inthe art may add appropriate pH adjusting ingredients to the compositionsof the present invention to adjust the pH to an acceptable range.“About” a specified pH is understood by those familiar with the art toinclude formulations in which at any given time the actual measured pHmay be less or more than the specified value by no more than 0.7, 0.6,0.5, 0.4., 0.3, 0.2 or 0.1 pH units, where it is recognized thatformulation composition and storage conditions may result in drifting ofpH from an original value.

A cream, lotion, gel, ointment, paste or the like may be spread on theaffected surface and gently rubbed in. A solution may be applied in thesame way, but more typically will be applied with a dropper, swab, orthe like, and carefully applied to the affected areas. The applicationregimen will depend on a number of factors that may readily bedetermined, such as the severity of the wound and its responsiveness toinitial treatment, but will normally involve one or more applicationsper day on an ongoing basis. One of ordinary skill may readily determinethe optimum amount of the formulation to be administered, administrationmethodologies and repetition rates. In general, it is contemplated thatthe formulations of these and related embodiments of the invention willbe applied in the range of once or twice or more weekly up to once,twice, thrice, four times or more daily.

As also discussed above, the topical formulations useful herein thusalso contain a pharmaceutically acceptable carrier, including anysuitable diluent or excipient, which includes any pharmaceutical agentthat does not itself harm the subject receiving the composition, andwhich may be administered without undue toxicity. Pharmaceuticallyacceptable carriers include, but are not limited to, liquids, such aswater, saline, glycerol and ethanol, and the like, and may also includeviscosity enhancers (e.g., balsam fir resin) or film-formers such ascolloidion or nitrocellulose solutions. A thorough discussion ofpharmaceutically acceptable carriers, diluents, and other excipients ispresented in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J.current edition).

When the topical formulation is in the form of a gel- or liquid-filledcapsule, for example, a gelatin capsule, it may contain, in addition tomaterials of the above type, a liquid carrier such as polyethyleneglycol or oil. The liquid pharmaceutical compositions of certainembodiments of the invention, whether they be solutions, suspensions orother like form, may include one or more of the following: sterilediluents such as water for injection, saline solution, preferablyphysiological saline, Ringer's solution, isotonic sodium chloride, fixedoils such as synthetic mono or diglycerides which may serve as thesolvent or suspending medium, polyethylene glycols, glycerin, propyleneglycol or other solvents; antibacterial agents such as benzyl alcohol ormethyl paraben; additional antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates and agents forthe adjustment of tonicity such as sodium chloride or dextrose.

For topical administration the carrier may suitably comprise a solution,emulsion, ointment or gel base. The base, for example, may comprise oneor more of the following: petrolatum, lanolin, polyethylene glycols, beewax, mineral oil, diluents such as water and alcohol, and emulsifiersand stabilizers. Thickening agents may be present in a pharmaceutical orcosmeceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device. Topical formulations may contain aconcentration of the compound of certain embodiments of the inventionfrom about 0.1 to about 10% w/v (weight per unit volume). A topicalformulation may be provided in the form of a cream, lotion, solution,spray, gel, ointment, paste or the like, and/or may contain liposomes,micelles, microspheres and/or other microparticle or nanoparticledelivery elements. A topical formulation may also be provided in theform of time-release or sustained release particles or pellets, forexample, slow-release ethylene vinyl acetate polymer (e.g., Elvax® 40,Aldrich, Milwaukee, Wis.) pellets, that can be directly administered toa wound site.

The topical formulation may include an agent that binds to the skintissue repair-promoting compound and thereby assists in its delivery toskin epithelial cells (e.g., keratinocytes) and/or fibroblasts. Suitableagents that may act in this capacity include clathrating agents such ascyclodextrins; other agents may include a protein or a liposome.

The topical formulation of certain embodiments of the invention may alsobe provided in the form of dosage units that can be administered as anaerosol. The term aerosol is used to denote a variety of systems rangingfrom those of colloidal nature to systems consisting of pressurizedpackages. Delivery may be by a liquefied or compressed gas or by asuitable pump system that dispenses the active ingredients. Aerosols ofcompounds of certain embodiments of the invention may be delivered insingle phase, bi-phasic, or tri-phasic systems in order to deliver theactive ingredient(s). Delivery of the aerosol includes the necessarycontainer, activators, valves, subcontainers, and the like, whichtogether may form a kit. One skilled in the art, without undueexperimentation may determine preferred aerosols for delivering topicalformulations to the skin or to a wound site.

The topical formulations may be prepared by methodology well known inthe pharmaceutical art. For example, a pharmaceutical compositionintended to be administered to a wound site or to the skin as a spray,wash or rinse can be prepared by combining a BTantiseptic/wound-healing/anti-biofilm/skin tissue repair-promotingcompound as described herein with sterile, distilled water so as to forma solution. A surfactant may be added to facilitate the formation of ahomogeneous solution or suspension. Surfactants are compounds thatnon-covalently interact with the antioxidant active compound so as tofacilitate dissolution or homogeneous suspension of the compound in theaqueous delivery system.

The BT antiseptic/wound-healing/anti-biofilm/skin tissuerepair-promoting compounds for use in topical formulations, or theirpharmaceutically acceptable salts, are administered in a therapeuticallyeffective amount, which will vary depending upon a variety of factorsincluding the nature of the wound site (where relevant), the activity ofthe specific BT compound employed (including the inclusion or absencefrom the formulation of an antibiotic, such as an aminoglycosideantibiotic, e.g., amikacin); the metabolic stability and length ofaction of the compound; the age, body weight, general health, sex, skintype, immune status and diet of the subject; the mode and time ofadministration; the rate of excretion; the drug combination; theseverity of the particular skin wound for which skin tissue repair isdesired; and the subject undergoing therapy. Generally, atherapeutically effective daily dose is (for a 70 kg mammal) from about0.001 mg/kg (i.e., 0.07 mg) to about 100 mg/kg (i.e., 7.0 g); preferablya therapeutically effective dose is (for a 70 kg mammal) from about 0.01mg/kg (i.e., 7 mg) to about 50 mg/kg (i.e., 3.5 g); more preferably atherapeutically effective dose is (for a 70 kg mammal) from about 1mg/kg (i.e., 70 mg) to about 25 mg/kg (i.e., 1.75 g).

The ranges of effective doses provided herein are not intended to belimiting and represent preferred dose ranges. However, the mostpreferred dosage will be tailored to the individual subject, as isunderstood and determinable by one skilled in the relevant arts. (see,e.g., Berkow et al., eds., The Merck Manual, 16^(th) edition, Merck andCo., Rahway, N.J., 1992; Goodman et al., eds., Goodman and Gilman's ThePharmacological Basis of Therapeutics, 10^(th) edition, Pergamon Press,Inc., Elmsford, N.Y., (2001); Avery's Drug Treatment: Principles andPractice of Clinical Pharmacology and Therapeutics, 3rd edition, ADISPress, Ltd., Williams and Wilkins, Baltimore, Md. (1987); Ebadi,Pharmacology, Little, Brown and Co., Boston, (1985); Osolci al., eds.,Remington's Pharmaceutical Sciences, 18^(th) edition, Mack PublishingCo., Easton, Pa. (1990); Katzung, Basic and Clinical Pharmacology,Appleton and Lange, Norwalk, Conn. (1992)).

The total dose required for each treatment can be administered bymultiple doses or in a single dose over the course of the day, ifdesired. Certain preferred embodiments contemplate a single applicationof the topical formulation per day. Generally, and in distinctembodiments, treatment may be initiated with smaller dosages, which areless than the optimum dose of the compound. Thereafter, the dosage isincreased by small increments until the optimum effect under thecircumstances is reached.

The topical formulation can be administered alone or in conjunction withother treatments and/or pharmaceuticals directed to the skin wound, ordirected to other associated symptoms or etiologic factors. For example,and as also noted above, the topical formulation may further compriseretinoic acid. As another example, the topical formulation may compriseone or more skin tissue repair-promoting compounds described herein, ormay comprise two or more such compounds having different cellular woundrepair activities.

The recipients of the topical formulations described herein can be anyvertebrate animal, such as mammals. Among mammals, the preferredrecipients are mammals of the Orders Primate (including humans, apes andmonkeys), Arteriodactyla (including horses, goats, cows, sheep, pigs),Rodenta (including mice, rats, rabbits, and hamsters), and Carnivora(including cats, and dogs). Among birds, the preferred recipients areturkeys, chickens and other members of the same order. The mostpreferred recipients are humans, and particularly preferred are humanshaving one or more acute or chronic wounds or wounds that containbiofilms.

For topical applications, it is preferred to administer an effectiveamount of a pharmaceutical composition comprising a BT compoundantiseptic/wound-healing/anti-biofilm/skin tissue repair-promotingcompound according to the herein described embodiments, to a targetarea, e.g., a skin wound such as an acute or chronic wound, and/or anat-risk area (e.g., for wound dehiscence) of the skin, and the like.This amount will generally range from about 0.0001 mg to about 1 g of acompound of certain embodiments of the invention per application,depending upon the area to be treated, the severity of the wound (or ofa past or contemplated surgical incision), and the nature of the topicalvehicle employed. A preferred topical preparation is an ointment orslow-release pellets, wherein about 0.001 to about 50 mg of activeingredient is used per cc of ointment base or pellet suspension. Thepharmaceutical composition can be formulated as transdermal compositionsor transdermal delivery devices (“patches”). Such compositions include,for example, a backing, active compound reservoir, a control membrane,liner and contact adhesive. Such transdermal patches may be used toprovide continuous pulsatile, or on demand delivery of the compounds ofthe present invention as desired.

The compositions of certain embodiments can be formulated so as toprovide quick, sustained or delayed release of the active ingredientafter administration to the patient by employing procedures known in theart. Controlled release drug delivery systems include osmotic pumpsystems and dissolutional systems containing polymer-coated reservoirsor drug-polymer matrix formulations. Examples of controlled releasesystems are given in U.S. Pat. Nos. 3,845,770 and 4,326,525 and in P. J.Kuzma et al, Regional Anesthesia 22 (6): 543-551 (1997), all of whichare incorporated herein by reference.

The most suitable route will depend on the nature and severity of thecondition being treated. Those skilled in the art are also familiar withdetermining topical administration methods (sprays, creams, openapplication, occlusive dressing, soaks, washes, etc.), dosage forms,suitable pharmaceutical excipients and other matters relevant to thedelivery of the compounds to a subject in need thereof.

Throughout this specification, unless the context requires otherwise,the words “comprise”, “comprises” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements. By “consisting of” is meant including, and limitedto, whatever follows the phrase “consisting of.” Thus, the phrase“consisting of” indicates that the listed elements are required ormandatory, and that no other elements may be present. By “consistingessentially of” is meant including any elements listed after the phrase,and limited to other elements that do not interfere with or contributeto the activity or action specified in the disclosure for the listedelements. Thus, the phrase “consisting essentially of” indicates thatthe listed elements are required or mandatory, but that no otherelements are required and may or may not be present depending uponwhether or not they affect the activity or action of the listedelements.

In this specification and the appended claims, the singular forms “a,”“an” and “the” include plural references unless the content clearlydictates otherwise. As used herein, in particular embodiments, the terms“about” or “approximately” when preceding a numerical value indicatesthe value plus or minus a range of 5%, 6%, 7%, 8% or 9%. In otherembodiments, the terms “about” or “approximately” when preceding anumerical value indicates the value plus or minus a range of 10%, 11%,12%, 13% or 14%. In yet other embodiments, the terms “about” or“approximately” when preceding a numerical value indicates the valueplus or minus a range of 15%, 16%, 17%, 18%, 19% or 20%.

The following Examples are presented by way of illustration and notlimitation.

EXAMPLES Example 1 Preparation of BT Compounds

The following BT compounds were prepared either according to the methodsof Domenico et al. (U.S. RE 37,793, U.S. Pat. No. 6,248,371, U.S. Pat.No. 6,086,921, U.S. Pat. No. 6,380,248) or as microparticles accordingto the synthetic protocol described below for BisEDT. Shown are atomicratios relative to a single bismuth atom, for comparison, based on thestoichiometric ratios of the reactants used and the known propensity ofbismuth to form trivalent complexes with sulfur containing compounds.The numbers in parenthesis are the ratios of bismuth to one (or more)thiol agents (e.g. Bi:thiol1/thiol2; see also Table 1).

 1) CPD 1B-1 Bis-EDT (1:1) BiC₂H₄S₂  2) CPD 1B-2 Bis-EDT (1:1.5)BiC₃H₆S₃  3) CPD 1B-3 Bis-EDT (1:1.5) BiC₃H₆S₃  4) CPD 1C Bis-EDT(soluble Bi prep.) (1:1.5) BiC₃H₆S₃  5) CPD 2A Bis-Bal (1:1) BiC₃H₆S₂O 6) CPD 2B Bis-Bal (1:1.5) BiC_(4.5)H₉O_(1.5)S₃  7) CPD 3A Bis-Pyr(1:1.5) BiC_(7.5)H₆N_(1.5)O_(1.5)S_(1.5)  8) CPD 3B Bis-Pyr (1:3)BiC₁₅H₁₂N₃O₃S₃  9) CPD 4 Bis-Ery (1:1.5) BiC₆H₁₂O₃S₃ 10) CPD 5 Bis-Tol(1:1.5) BiC_(10.5)H₉S₃ 11) CPD 6 Bis-BDT (1:1.5) BiC₆H₁₂S₃ 12) CPD 7Bis-PDT (1:1.5) BiC_(4.5)H₉S₃ 13) CPD 8-1 Bis-Pyr/BDT (1:1/1) 14) CPD8-2 Bis-Pyr/BDT (1:1/0.5) 15) CPD 9 Bis-2hydroxy, propane thiol (1:3)16) CPD 10 Bis-Pyr/Bal (1:1/0.5) 17) CPD 11 Bis-Pyr/EDT (1:1/0.5) 18)CPD 12 Bis-Pyr/Tol (1:1/0.5) 19) CPD 13 Bis-Pyr/PDT (1:1/0.5) 20) CPD 14Bis-Pyr/Ery (1:1/0.5) 21) CPD 15 Bis-EDT/2hydroxy, propane thiol (1:1/1)

Microparticulate bismuth-1,2-ethanedithiol (Bis-EDT, soluble bismuthpreparation) was prepared as follows:

To an excess (11.4 L) of 5% aqueous HNO₃ at room temperature in a 15 Lpolypropylene carboy was slowly added by dropwise addition 0.331 L(˜0.575 moles) of an aqueous Bi(NO₃)₃ solution (43% Bi(NO₃)₃ (w/w), 5%nitric acid (w/w), 52% water (w/w), Shepherd Chemical Co., Cincinnati,Ohio, product no. 2362; δ˜1.6 g/mL) with stirring, followed by slowaddition of absolute ethanol (4 L). Some white precipitate formed butwas dissolved by continued stirring. An ethanolic solution (˜1.56 L,˜0.55 M) of 1,2-ethanedithiol (CAS 540-63-6) was separately prepared byadding, to 1.5 L of absolute ethanol, 72.19 mL (0.863 moles) of1,2-ethanedithiol using a 60 mL syringe, and then stirring for fiveminutes. The 1,2-ethanedithiol/EtOH reagent was then slowly added bydropwise addition over the course of five hours to the aqueousBi(NO₃)₃/HNO₃ solution, with continued stirring overnight. The formedproduct was allowed to settle as a precipitate for approximately 15minutes, after which the filtrate was removed at 300 mL/min using aperistaltic pump. The product was then collected by filtration on finefilter paper in a 15-cm diameter Buchner funnel, and washed sequentiallywith three, 500-mL volumes each of ethanol, USP water, and acetone toobtain BisEDT (694.51 gm/mole) as a yellow amorphous powdered solid. Theproduct was placed in a 500 mL amber glass bottle and dried over CaCl₂under high vacuum for 48 hours. Recovered material (yield ˜200 g) gaveoff a thiol-characteristic odor. The crude product was redissolved in750 mL of absolute ethanol, stirred for 30 min, then filtered and washedsequentially with 3×50 mL ethanol, 2×50 mL acetone, and washed againwith 500 mL of acetone. The rewashed powder was triturated in 1M NaOH(500 mL), filtered and washed with 3×220 mL water, 2×50 mL ethanol, and1×400 mL acetone to afford 156.74 gm of purified BisEDT. Subsequentbatches prepared in essentially the same manner resulted in yields ofabout 78-91%.

The product was characterized as having the structure shown above informula I by analysis of data from ¹H and ¹³C nuclear magnetic resonance(NMR), infrared spectroscopy (IR), ultraviolet spectroscopy (UV), massspectrometry (MS) and elemental analysis. An HPLC method was developedto determine chemical purity of BisEDT whereby the sample was preparedin DMSO (0.5 mg/mL). The λ_(max) was determined by scanning a solutionof BisEDT in DMSO between 190 and 600 nm. Isocratic HPLC elution at 1mL/min was performed at ambient temperature in a mobile phase of 0.1%formic acid in acetonitrile:water (9:1) on a Waters (Millipore Corp.,Milford, Mass.) model 2695 chromatograph with UV detector monitoring at265 nm (λ_(max)), 2 μL injection volume, equipped with a YMC Pack PVCSil NP, 5 μm, 250×4.6 mm inner diameter analytical column (Waters) and asingle peak was detected, reflecting chemical purity of 100±0.1%.Elemental analysis was consistent with the structure of formula (I).

The dried particulate matter was characterized to assess the particlesize properties. Briefly, microparticles were resuspended in 2%Pluronic® F-68 (BASF, Mt. Olive, N.J.) and the suspension was sonicatedfor 10 minutes in a water bath sonicator at standard setting prior toanalysis using a Nanosizer/Zetasizer Nano-S particle analyzer (modelZEN1600 (without zeta-potential measuring capacity), MalvernInstruments, Worcestershire, UK) according to the manufacturer'srecommendations. From compiled data of two measurements, microparticlesexhibited a unimodal distribution with all detectable events betweenabout 0.6 microns and 4 microns in volumetric mean diameter (VMD) andhaving a peak VMD at about 1.3 microns. By contrast, when BisEDT wasprepared by prior methods (Domenico et al., 1997 Antimicrob. AgentsChemother. 41(8):1697-1703) the majority of particles wereheterodisperse and of significantly larger size, precluding theircharacterization on the basis of VMD.

Example 2 Colony Biofilm Model of Chronic Wound Infection: Inhibition byBT Compounds

Because bacteria that exist in chronic wounds adopt a biofilm lifestyle,BTs were tested against biofilms for effects on bacterial cell survivalusing biofilms prepared essentially according to described methods(Anderl et al., 2003 Antimicrob Agents Chemother 47:1251-56; Walters etal., 2003 Antimicrob Agents Chemother 47:317; Wentland et al., 1996Biotchnol. Prog. 12:316; Zheng et al., 2002 Antimicrob Agents Chemother46:900).

Briefly, colony biofilms were grown on 10% tryptic soy agar for 24hours, and transferred to Mueller Hinton plates containing treatments.After treatment the biofilms were dispersed into peptone watercontaining 2% w/v glutathione (neutralizes the BT), and serially dilutedinto peptone water before being spotted onto plates for counting. Twobacteria isolated from chronic wounds were used separately in theproduction of colony biofilms for testing. These were Pseudomonasaeruginosa, a gram negative bacterial strain, and Methicillin ResistantStaphylococcus aureus (MRSA), which is gram positive.

Bacterial biofilm colonies were grown on top of micro porous membranesresting on an agar plate essentially as described (Anderl et al., 2003Antimicrob Agents Chemother 47:1251-56; Walters et al., 2003 AntimicrobAgents Chemother 47:317; Wentland et al., 1996 Biotchnol. Prog. 12:316;Zheng et al., 2002 Antimicrob Agents Chemother 46:900) The colonybiofilms exhibited many of the familiar features of other biofilmmodels, e.g., they consisted of cells densely aggregated in a highlyhydrated matrix. As also reported by others (Brown et al., J Surg Res56:562; Millward et al, 1989 Microbios 58:155; Sutch et al., 1995 JPharm Pharmacol 47:1094; Thrower et al., 1997 J Med Microbiol 46:425) itwas observed that bacteria in colony biofilms exhibited the sameprofoundly reduced anti-microbial susceptibility that has beenquantified in more sophisticated in vitro biofilm reactors. Colonybiofilms were readily and reproducibly generated in large numbers.According to non-limiting theory, this colony biofilm model shared someof the features of an infected wound: bacteria grew at an air interfacewith nutrients supplied from beneath the biofilm and minimal fluid flow.A variety of nutrients sources was used to cultivate colony biofilms,including blood agar, which is believed to mimic in vivo nutrientconditions.

Colony biofilms were prepared by inoculating 5 μl spots of planktonicbacterial liquid cultures onto a 25 mm diameter polycarbonate filtermembrane. The membranes were sterilized prior to inoculation, byexposure to ultraviolet light for 10 min per side. The inocula weregrown overnight in bacterial medium at 37° C. and diluted in freshmedium to an optical density of 0.1 at 600 nm prior to deposition on themembrane. The membranes were then placed on the agar plate containinggrowth medium. The plates were then covered and placed, inverted, in anincubator at 37° C. Every 24 h, the membrane and colony biofilm weretransferred, using sterile forceps, to a fresh plate. Colony biofilmswere typically used for experimentation after 48 hours of growth, atwhich time there were approximately 10⁹ bacteria per membrane. Thecolony biofilm method was successfully employed to culture a widevariety of single species and mixed species biofilms.

To measure susceptibility to antimicrobial agents (e.g., BT compoundsincluding combinations of BT compounds; antibiotics; and BTcompound-antibiotic combinations), colony biofilms were transferred toagar plates supplemented with the candidate antimicrobial treatmentagent(s). Where the duration of exposure to antimicrobial treatmentexceeded 24 hours, the colony biofilms were moved to fresh treatmentplates daily. At the end of the treatment period, the colony biofilmswere placed in tubes containing 10 ml of buffer and vortexed for 1-2 minto disperse the biofilm. In some cases, it was necessary to brieflyprocess the sample with a tissue homogenizer to break up cellaggregates. The resulting cell suspensions were then serially dilutedand plated to enumerate surviving bacteria, which were reported ascolony forming units (CFU) per unit area. Survival data were analyzedusing log₁₀ transformation.

For each type of bacterial biofilm colony cultures (Pseudomonasaeruginosa, PA; methicilin resistant Staphylococcus aureus, MRSA or SA)five antibiotics and thirteen BT compounds were tested. Antimicrobialagents tested against PA included the BTs referred to herein as BisEDTand Compounds 2B, 4, 5, 6, 8-2, 9, 10, 11 and 15 (see Table 1), and theantibiotics tobramycin, amikacin, imipenim, cefazolin, andciprofloxacin. Antimicrobial agents tested against SA included the BTsreferred to herein as BisEDT and Compounds 2B, 4, 5, 6, 8-2, 9, 10 and11 (see Table 1), and the antibiotics rifampicin, daptomycin,minocycline, ampicillin, and vancomycin. As described above under “briefdescriptions of the drawings”, antibiotics were tested at concentrationsof approximately 10-400 times the minimum inhibitory concentrations(MIC) according to established microbiological methodologies.

Seven BT compounds exhibited pronounced effects on PA bacterial survivalat the concentrations tested, and two BT compounds demonstratedpronounced effects on MRSA survival at the concentrations tested;representative results showing BT effects on bacterial survival arepresented in FIG. 1 for BisEDT and BT compound 2B (tested against PA)and in FIG. 2 for BT compounds 2B and 8-2 (tested against SA), in bothcases, relative to the effects of the indicated antibiotics. As alsoshown in FIGS. 1 and 2, inclusion of the indicated BT compounds incombination with the indicated antibiotics resulted in a synergisticeffect whereby the potency of reducing bacterial survival was enhancedrelative to the anti-bacterial effects of either the antibiotic alone orthe BT compound alone. In the PA survival assay, compound 15(Bis-EDT/2hydroxy, propane thiol (1:1/1)) at a concentration of 80 μg/mLexhibited an effect (not shown) that was comparable to the effectobtained using the combination of 1600 μg/mL AMK plus 80 μg/mL BisEDT(FIG. 1).

Example 3 Drip Flow Biofilm Model of Chronic Wound Infection: Inhibitionby BT Compounds

Drip flow biofilms represent an art accepted authentic model forforming, and testing the effect of candidate anti-bacterial compoundsagainst, bacterial biofilms. Drip flow biofilms are produced on coupons(substrates) placed in the channels of a drip flow reactor. Manydifferent types of materials can be used as the substrate for bacterialbiofilm formation, including frosted glass microscope slides. Nutritiveliquid media enters the drip flow bioreactor cell chamber by drippinginto the chamber near the top, and then flows the length of a coupondown a 10 degree slope.

Biofilms are grown in drip flow bioreactors and exposed to BT compoundsindividually or in combinations and/or to antibiotic compoundsindividually or in combinations with other antibacterial agents,including BT compounds, or to other conventional or candidate treatmentsfor chronic wounds. BT compounds are thus characterized for theireffects on bacterial biofilms in the drip-flow reactor. Biofilms in thedrip-flow reactor are prepared according to established methodologies(e.g., Stewart et al., 2001 J Appl Microbiol. 91:525; Xu et al., 1998Appl. Environ. Microbiol. 64:4035). This design involves cultivatingbiofilms on inclined polystyrene coupons in a covered chamber. Anexemplary culture medium contains 1 g/l glucose, 0.5 g/l NH₄NO₃, 0.25g/l KCl, 0.25 g/l KH₂PO₄, 0.25 g/l MgSO₄-7H₂O, supplemented with 5% v/vadult donor bovine serum (ph 6.8) that mimics serum protein-rich, ironlimited conditions that are similar to biofilm growth conditions invivo, such as in chronic wounds. This medium flows drop-wise (50 ml/h)over four coupons contained in four separate parallel chambers, each ofwhich measures 10 cm×1.9 cm by 1.9 cm deep. The chambered reactor isfabricated from polysulfone plastic. Each of the chambers is fitted withan individual removable plastic lid that can be tightly sealed. Thebiofilm reactor is contained in an incubator at 37° C., and bacterialcell culture medium is warmed by passing it through an aluminum heatsink kept in the incubator. This method reproduces the antibiotictolerant phenotype observed in certain biofilms, mimics the low fluidshear environment and proximity to an air interface characteristic of achronic wound while providing continual replenishment of nutrients, andis compatible with a number of analytical methods for characterizing andmonitoring the effects of introduced candidate antibacterial regimens.The drip-flow reactor has been successfully employed to culture a widevariety of pure and mixed-species biofilms. Biofilms are typically grownfor two to five days prior to application of antimicrobial agents.

To measure the effects of anti-biofilm agents on biofilms grown indrip-flow reactors, the fluid stream passing over the biofilm is amendedor supplemented with the desired treatment formulation (e.g., one ormore BT compounds and/or one or more antibiotics, or controls, and/orother candidate agents). Flow is continued for the specified treatmentperiod. The treated biofilm coupon is then briefly removed from thereactor and the biofilm is scraped into a beaker containing 10 ml ofbuffer. This sample is briefly processed (typically 30 s to 1 min) witha tissue homogenizer to disperse bacterial aggregates. The suspension isserially diluted and plated to enumerate surviving microorganismsaccording to standard microbiological methodologies.

Example 4 Wound Biofilm Inhibition of Keratinocyte Scratch Repair:Biofilm Suppression by BT Compounds

This Example describes a modification of established in vitrokeratinocyte scratch models of wound healing, to arrive at a modelhaving relevance to biofilm-associated wound pathology and woundhealing, and in particular to acute or chronic wounds or woundscontaining biofilms as described herein. According to the keratinocytescratch model of the effects of chronic wound biofilms, cultivation ofmammalian (e.g., human) keratinocytes and bacterial biofilm populationsproceeds in separate chambers that are in fluid contact with oneanother, to permit assessment of the effects of conditions thatinfluence the effects, of soluble components elaborated by biofilms, onkeratinocyte wound healing events.

Newborn human foreskin cells are cultured as monolayers in treatedplastic dishes, in which monolayers a controlled “wound” or scratch isformed by mechanical means (e.g., through physical disruption of themonolayer such as by scraping an essentially linear cell-free zonebetween regions of the monolayer with a suitable implement such as asterile scalpel, razor, cell scraper, forceps or other tool). In vitrokeratinocyte monolayer model systems are known to undergo cellularstructural and functional process in response to the wounding event, ina manner that simulates wound healing in vivo. According to the hereindisclosed embodiments, the influence of the presence of bacterialbiofilms on such processes, for instance, on the healing time of thescratch, is observed, and in these and related embodiments the effectsare also assessed of the presence of selected candidate antimicrobial(e.g., antibacterial and antibiofilm) treatments.

Wounded keratinocyte monolayers cultured in the presence of biofilms areexamined according to morphological, biochemical, molecular genetic,cell physiologic and other parameters to determine whether introductionof BT comopunds alters (e.g., increases or decreases in a statisticallysignificant manner relative to appropriate controls) the damagingeffects of the biofilms. Wounds are first exposed to each BT compoundalone, and to contemplated combinations of BT compounds, in order totest the toxicity of each BT compound treatment prior to assessing theeffects of such treatments on biofilm influences toward the model woundhealing process.

In a representative embodiment, a three-day biofilm is cultured on amembrane (e.g., a TransWell membrane insert or the like) that ismaintained in a tissue culture well above, and in fluid communicationwith, a keratinocyte monolayer that is scratched to initiate the woundhealing process. Biofilms cultured out of authentic acute or chronicwounds are contemplated for use in these and related embodiments.

Thus, an in vitro system has been developed for evaluating solublebiofilm component effects on migration and proliferation of humankeratinocytes. The system separates the biofilm and keratinocytes usinga dialysis membrane. Keratinocytes are cultured from newborn foreskin aspreviously described (Fleckman et al., 1997 J Invest. Dermatol. 109:36;Piepkorn et al., 1987 J Invest. Dermatol. 88:215-219) and grown asconfluent monolayers on glass cover slips. The keratinocyte monolayerscan then be scratched to yield “wounds” with a uniform width, followedby monitoring cellular repair processes (e.g., Tao et al., 2007 PLoS ONE2:e697; Buth et al. 2007 Eur. J Cell Biol. 86:747; Phan et al. 2000 Ann.Acad. Med. Singapore 29:27). The artificial wounds are then placed inthe bottom of a sterile double-sided chamber and the chamber isassembled using aseptic technique. Both sides of the chamber are filledwith keratinocyte growth medium (EpiLife) with or without antibioticsand/or bismuth-thiols. Uninoculated systems are used as controls.

The system is inoculated with wound-isolated bacteria and incubated instatic conditions for two hours to enable bacterial attachment tosurfaces in the upper chambers. Following the attachment period, liquidmedium flow is initiated in the upper chamber to remove unattachedcells. Flow of medium is then continued at a rate that minimizes thegrowth of planktonic cells within the upper chamber, by washout ofunattached cells. After incubation periods ranging from 6 to 48 hours,the systems (keratinocyte monolayers on coverslips and bacterial biofilmon membrane substrate) are disassembled and the cover slips removed andanalyzed. In related embodiments, mature biofilms are grown in the upperchamber prior to assembling the chamber. In other related embodiments,the separate co-culturing of biofilms and scratch-wounded keratinocytemonolayers is conducted in the absence and presence of one or more BTcompounds, optionally with the inclusion or exclusion of one or moreantibiotics, in order to determine effects of candidate agents such asBT compounds, or of potentially synergizing BT compound-plus-antibioticcombinations (e.g., a BT compound as provided herein such as a BT thatis provided in microparticulate form, and one or more of amikacin,ampicillin, cefazolin, cefepime, chloramphenicol, ciprofloxacin,clindamycin (or another lincoasamide antibiotic), daptomycin (Cubicin®),doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin, linezolid(Zyvox®), minocycline, nafcilin, paromomycin, rifampin,sulphamethoxazole, tobramycin and vancomycin), on keratinocyte repair ofthe scratch wound, e.g., to identify an agent or combination of agentsthat alters (e.g., increases or decreases in a statistically significantmanner relative to appropriate controls) at least one indicator ofscratch wound healing, such as the time elapsing for wound repair totake place or other wound-repair indicia (e.g., Tao et al., 2007 PLoSONE 2:e697; Buth et al. 2007 Eur. J Cell Biol. 86:747; Phan et al. 2000Ann. Acad. Med. Singapore 29:27).

Example 5 Wound Biofilm Inhibition of Keratinocyte Scratch Repair

Isolated human keratinocytes were cultured on glass coverslips andscratch-wounded according to methodologies described above in Example 4.Wounded cultures were maintained under culture conditions alone or inthe presence of a co-cultured biofilm on a membrane support in fluidcommunication with the keratinocyte culture. The scratch closure timeinterval during which keratinocyte cell growth and/or migrationreestablishes the keratinocyte monolayer over the scratch zone was thendetermined. FIG. 3 illustrates the effect that the presence in fluidcommunication (but without direct contact) of biofilms had on thehealing time of scratched keratinocyte monolayers.

Accordingly there are contemplated in certain embodiments a method ofidentifying an agent for treating a chronic wound, comprising culturinga scratch-wounded cell (e.g., keratinocyte or fibroblast) monolayer inthe presence of a bacterial biofilm with and without a candidateanti-biofilm agent being present; and assessing an indicator of healingof the scratch-wounded cell monolayer in the absence and presence of thecandidate anti-biofilm agent, wherein an agent (e.g., a BT compound suchas a substantially monodisperse BT microparticle suspension as describedherein, alone or in synergizing combination with an antibiotic, such asone or more of amikacin, ampicillin, cefazolin, cefepime,chloramphenicol, ciprofloxacin, clindamycin, daptomycin (Cubicin®),doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin, linezolid(Zyvox®), minocycline, nafcilin, paromomycin, rifampin,sulphamethoxazole, tobramycin and vancomycin) that promotes at least oneindicator of healing is identified as a suitable agent for treating anacute or chronic wound or a wound that contains a biofilm.

Example 6 Synergizing Bismuth-Thiol (BT)-Antibiotic Combinations

This example shows instances of demonstrated synergizing effects bycombinations of one or more bismuth-thiol compounds and one or moreantibiotics against a variety of bacterial species and bacterialstrains, including several antibiotic-resistant bacteria.

Materials & Methods. Susceptibility studies were performed by brothdilution in 96-well tissue culture plates (Nalge Nunc International,Denmark) in accordance with NCCLS protocols (National Committee forClinical Laboratory Standards. (1997). Methods for DilutionAntimicrobial Susceptibility Tests for Bacteria that Grow Aerobically:Approved Standard M7-A2 and Informational Supplement M100-S10. NCCLS,Wayne, Pa., USA).

Briefly, overnight bacterial cultures were used to prepare 0.5 McFarlandstandard suspensions, which were further diluted 1:50 (˜2×10⁶ cfu/mL) incation-adjusted Mueller-Hinton broth medium (BBL, Cockeysville, Md.,USA). BTs (prepared as described above) and antibiotics were added atincremental concentrations, keeping the final volume constant at 0.2 mL.Cultures were incubated for 24 h at 37° C. and turbidity was assessed byabsorption at 630 nm using an ELISA plate reader (Biotek Instruments,Winooski, Vt., USA) according to the manufacturer's recommendations. TheMinimum Inhibitory Concentration (MIC) was expressed as the lowest drugconcentration inhibiting growth for 24 h. Viable bacterial counts(cfu/mL) were determined by standard plating on nutrient agar. TheMinimal Bactericidal Concentrations (MBC) was expressed as theconcentration of drug that reduced initial viability by 99.9% at 24 h ofincubation.

The checkerboard method was used to assess the activity of antimicrobialcombinations. The fractional inhibitory concentration index (FICI) andthe fractional bactericidal concentration index (FBCI) were calculated,according to Eliopoulos et al. (Eliopoulos and Moellering, (1996)Antimicrobial combinations. In Antibiotics in Laboratory Medicine(Lorian, V., Ed.), pp. 330-96, Williams and Wilkins, Baltimore, Md.,USA). Synergy was defined as an FICI or FBCI index of ≤0.5, nointeraction at >0.5-4 and antagonism at >4 (Odds, FC (2003) Synergy,antagonism, and what the chequerboard puts between them. Journal ofAntimicrobial Chemotherapy 52:1). Synergy was also definedconventionally as ≥4-fold decrease in antibiotic concentration.

Results are presented in Tables 2-17.

TABLE 2 S. aureus Nafcilin resistant NAF/BE NAF MIC MIC Strain (μg/ml)(μg/ml) Δ Synergy 60187-2 10.00 0.6 16.7 + 52446-3 175.00 40.0 4.4 +M1978 140.00 50.0 2.8 − W54793 130.00 33.3 3.9 − S24341 210.00 65.0 3.2− H7544 28.33 15.0 1.9 − H72751 145.00 43.3 3.3 − W71630 131.67 46.7 2.8− X22831 178.33 75.0 2.4 − X23660 123.33 43.3 2.8 − O36466 191.67 93.32.1 BE = 0.2 μg/ml BisEDT; Bacterial strains were obtained from theClinical Microbiology Laboratory at Winthrop-University Hospital,Mineola, NY. Nafcillin was obtained from Sigma (St. Louis, MO).

TABLE 3 S. aureus Nafcilin resistant GM/BE GM MIC MIC Strain (μg/ml)(μg/ml) Δ Synergy 60187-2 0.233 0.004 58.3 + 52446-3 10.667 1.500 7.1 +M1978 32.500 4.000 8.1 + W54793 0.250 0.080 3.1 − S24341 0.250 0.0584.3 + H7544 0.383 0.093 4.1 + H72751 0.200 0.072 2.8 − W71630 17.6673.800 4.6 + X22831 − 0.085 X23660 22.500 4.000 5.6 + O36466 0.267 0.0436.2 + BE = 0.2 μg/ml BisEDT; Bacterial strains were obtained from theClinical Microbiology Laboratory at Winthrop-University Hospital,Mineola, NY. Nafcillin was obtained from Sigma.

TABLE 4 S. aureus Rifampin/Neomycin/Paromomycin MIC MIC + BE (μg/ml)(μg/ml) Δ Synergy ATCC 25923 RIF 0.033 0.003 13.0 + NEO 0.500 0.200 2.5− PARO 1.080 0.188 5.7 + MRSA S2446-3 RIF 2.500 2.500 1.0 − NEO 13.4008.500 1.6 − PARO 335.000 183.300 1.8 − BE = 0.2 μg/ml BisEDT; StrainS2446-3 was obtained from the Clinical Microbiology Laboratory atWinthrop-University Hospital, Mineola, NY. Antibiotics were obtainedfrom Sigma.

TABLE 5 S. epidermidis - GM resistant strain ATCC 35984 strain S2400-1BisEDT MIC MBC MIC MBC (μg/ml) (μg/ml GM) (μg/ml GM) (μg/ml GM) (μg/mlGM) 0 53.3 384.0 85.3 426.7 0.005 20.0 96.0 96.0 512.0 0.01 37.3 117.364.0 256.0 0.02 21.3 26.7 28.0 128.0 0.04 2.0 16.0 2.0 128.0 0.08 2.010.7 2.0 53.3 0.16 (MIC) 3.0 10.0 0.32 2.0 4.0 GM = gentamicin; StrainS2400-1 was obtained from the Clinical Microbiology Laboratory atWinthrop-University Hospital, Mineola, NY. Gentamicin was obtained fromthe Pharmacy Department at Winthrop; synergy in bold

TABLE 6 S. epidermidis - S2400-1 Biofilm Prevention BisEDT (μg/ml) ΔAntibiotic 0 0.05 0.1 (0.05 BE) Synergy cefazolin 28 10 1 2.8 −vancomycin 3.2 0.9 0.1 3.6 − gatifloxacin 1.6 0.1 0.1 16.0 ++ rifampicin0.03 0.04 0.04 0.7 − nafcillin 48 64 8 0.8 − clindamycin 1195 48 12 24.9++++ gentamicin 555 144 12 3.9 borderline minocycline 0.85 0.73 0.08 1.2− Data in μg/ml; Strain S2400-1 was obtained from the ClinicalMicrobiology Laboratory at Winthrop-University Hospital, Mineola, NY.Antibiotics were obtained from the Pharmacy Department at Winthrop.

TABLE 7 S. epidermidis - S2400-1 MIC BisEDT (μg/ml) Δ Antibiotic 0 0.050.1 (0.05 BE) Synergy cefazolin 32 8 1 4.00 + vancomycin 3.2 2.3 0.31.40 − gatifloxacin 1.7 0.8 0.3 2.13 − rifampicin 0.03 0.04 0.04 0.75 −nafcillin 171 192 68 0.89 − clindamycin 2048 768 24 2.67 − gentamicin2048 320 80 6.40 + minocycline 1.13 0.43 0.10 2.63 − Data in μg/ml;Strain S2400-1 was obtained from the Clinical Microbiology Laboratory atWinthrop-University Hospital, Mineola, NY. Antibiotics were obtainedfrom the Pharmacy Department at Winthrop.

TABLE 8 S. epidermidis - S2400-1 MBC BisEDT (μg/ml) Δ Antibiotic 0.0 0.1(0.1 BE) Synergy cefazolin 48 10 4.80 + vancomycin 5.4 1.4 3.86borderline gatifloxacin 2.8 1.4 2.00 − rifampicin 0.03 0.07 0.43 −nafcillin 256 128 2.00 − clindamycin 2048 768 2.67 − gentamicin 1536 2566.00 + minocycline 1.20 1.20 1.00 − Data in μg/ml; Strain S2400-1 wasobtained from the Clinical Microbiology Laboratory atWinthrop-University Hospital, Mineola, NY. Antibiotics were obtainedfrom the Pharmacy Department at Winthrop.

TABLE 9 S. epidermidis ATCC 35984 MIC BisEDT (μg/ml) Antibiotic 0.0 0.05Δ Synergy Nafcillin 16.00 5.00 3.2 − Clindamycin 2048.00 1024.00 2 −Gentamicin 213.33 16.00 13.3 ++ Minocycline 0.13 0.04 3.3 − Rifampicin0.021 0.014 1.5 − Data in μg/ml; Antibiotics were obtained from thePharmacy Department at Winthrop-University Hospital, Mineola, NY.

TABLE 10 E. coli - Ampicillin/Chloramphenicol resistant MIC MIC AB AB/BEMIC BE Strain (μg/ml) (μg/ml AB) Δ Synergy (μg/ml) MC4100/TN9 220 12.717.4 + 0.6 (CM) MC4100/P9 285 49 5.8 + 0.5 (AM) MC4100 (AM) 141.7 354.0 + 0.6 AB = antibiotic; CM = chloramphenicol; AM = ampicillin; BE =BisEDT at 0.3 μg/ml; Strains were obtained from the laboratory of Dr. MJ Casadaban, Department of Molecular Genetics and Cell Biology, TheUniversity of Chicago, Chicago, IL. Antibiotics were obtained from thePharmacy Department at Winthrop-University Hospital, Mineola, NY.

TABLE 11 E. coli - Tetracycline-resistant: Doxycycline + BisEDT DOX MICDOX/BE MIC BE MIC Strain (μg/ml) (μg/ml DOX) Δ Synergy (μg/ml) TET M16.50 4.50 4.0 + 0.85 TET D 20.50 0.03 820.0 ++++ 0.85 TET A 15.00 10.001.5 − 0.40 TET B 20.13 10.33 2.0 − 0.60 DOX = doxycycline; BE = BisEDTat 0.3 μg/ml; Strains were obtained from the laboratory of Dr. I Chopra,Department of Bacteriology, The University of Bristol, Bristol, UK.Antibiotics were obtained from the Pharmacy Department atWinthrop-University Hospital, Mineola, NY.

TABLE 12 P. aeruginosa - Tobramycin-resistant: BisEDT Synergy NN NN + BEBE MIC Strain (μg/ml) (μg/ml NN) Δ Synergy (μg/ml) Xen5 0.32 0.19 1.68 −0.9 Agr PA E 115 70 1.64 − 0.9 Agr PA I 200 73 2.74 − 1 Agr PA K 4.8 31.60 − 0.82 Agr PA O 130 20.5 6.34 + 0.98 Agr = aminoglycosideresistant; NN = tobramycin; PA = Pseudomonas aeruginosa; BE = BisEDT,0.3 μg/ml; Strains were obtained from the laboratory of Dr. K. Poole,Department of Microbiology and Immunology, Queens University, Ontario,CN. Tobramycin was obtained from the Pharmacy Department atWinthrop-University Hospital, Mineola, NY.

TABLE 13 B. cepacia Tobramycin + BE Synergy MIC NN NN + BE BE MIC Strain(μg/ml) (μg/ml NN) Δ Synergy (μg/ml) 13945 200 50 4 + 2.4 25416 125 1012.5 ++ 1.2 HI 2229 64 8 8 + 0.8 AU 0267 128 2 64 ++++ 0.8 AU 0259 1024256 4 + 1.6 HI 2255 64 8 8 + 1.6 AU 0273 512 32 16 ++ 1.6 HI 2253 64 164 + 1.6 HI 2147 512 8 64 ++++ 1.6 NN = Tobramycin; BE = BisEDT, 0.4μg/ml; Strains were obtained from the laboratory of Dr. J. J. LiPuma,Department of Pediatrics and Communicable Diseases, University ofMichigan, Ann Arbor, MI; also Veloira et al. 2003. Tobramycin wasobtained from the Pharmacy Department at Winthrop-University Hospital,Mineola, NY.

TABLE 14 B. cepacia Tobramvcin + BE Synergy MBC NN NN + BE BE MIC Strain(μg/ml) (μg/ml NN) Δ Synergy (μg/ml) HI 2249 256 8 32 ++ 3.2 HI 2229 12832 4 + 6.4 AU 0267 256 32 8 + 6.4 AU 0259 1024 1024 1 − 12.8 HI 2255 12832 4 + 12.8 HI 2711 512 8 64 ++++ 6.4 AU 0284 1024 64 16 ++ 0.8 AU 0273512 32 16 ++ 1.6 HI 2253 128 64 2 − 3.2 HI 2147 512 128 4 + 6.4 NN =Tobramycin; BE = BisEDT, 0.4 μg/ml; Strains were obtained from thelaboratory of Dr. J. J. LiPuma, Department of Pediatrics andCommunicable Diseases, University of Michigan, Ann Arbor, MI; alsoVeloira et al. 2003. Tobramycin was obtained from the PharmacyDepartment at Winthrop-University Hospital, Mineola, NY.

TABLE 15 Tobramycin Resistant Strains MIC NN NN + BE Lipo-BE-NN Strain(μg/ml) (μg/ml NN) Δ Synergy (μg/ml NN) M13637 512 32 16 ++ 0.25 M13642R128 64 2 − 0.25 PA-48913 1024 256 4 + 0.25 PA-48912-2 64 8 8 + 0.25PA-10145 1 4 0.25 − 0.25 SA-29213 2 1 2 − 0.25 NN = Tobramycin; BE =BisEDT, 0.8 μg/ml; Lipo-BE-NN = liposomal BE-NN; Strains were obtainedfrom the laboratory of Dr. A. Omri, Department of Chemistry andBiochemistry, Laurentian University, Ontario, CN; (M strains are mucoidB. cepacia; PA = P. aeruginosa; SA = S. aureus). Tobramycin was obtainedfrom the Pharmacy Department at Winthrop-University Hospital, Mineola,NY.

TABLE 16 Tobramycin Resistant Strains MBC NN NN + BE Lipo-BE-NN Strain(μg/ml) (μg/ml NN) Δ Synergy (μg/ml NN) M13637 1024 64 16 ++ 8 M13642R256 128 2 − 16 PA-48913 4096 512 8 + 4 PA-48912-2 128 32 4 + 0.5PA-10145 1 8 0.125 − 4 SA-29213 2 1 2 − 0.25 NN = Tobramycin; BE =BisEDT, 0.8 μg/ml; Lipo-BE-NN = liposomal BE-NN; Strains were obtainedfrom the laboratory of Dr. A. Omri, Department of Chemistry andBiochemistry, Laurentian University, Ontario, CN; (M strains are mucoidB. cepacia; PA = P. aeruginosa; SA = S. aureus). Tobramycin was obtainedfrom the Pharmacy Department at Winthrop-University Hospital, Mineola,NY.

TABLE 17 BisEDT-Pyrithione Synergy P. aeruginosa E. coli S. aureus NaPYRATCC 27853 ATCC 25922 ATCC 25923 (ug/ml) (μg/ml BE) (μg/ml BE) (μg/mlBE) 0 0.25 0.1 0.25 0.025 0.1 0.125 0.05 0.025 0.063 0.1 0.125 0.01250.063 0.2 0.125 0.0125 0.031 0.4 0.00625 0 0.8 0.125 0.00625 1.6 (MIC)0.063 0.00625 3.2 0.063 0 6.4 0.063 12.8 0 BE = BisEDT; NaPYR = sodiumpyrithione; Chemicals were obtained from Sigma-Aldrich; synergy in bold.Indicated bacterial strains were from American Type Culture Collection(ATCC, Manassas, VA).

Example 7 Comparative Bismuth-Thiol (BT) and Antibiotic Effects AgainstGram-Positive and Gram-Negative Bacteria Including Antibiotic-ResistantBacterial Strains

In this example the in vitro activities of BisEDT and comparator agentswere assessed against multiple clinical isolates of Gram-positiveand—negative bacteria that are responsible for skin and soft tissueinfections.

Materials and Methods. Test compounds and test concentration ranges wereas follows: BisEDT (Domenico et al., 1997; Domenico et al., Antimicrob.Agents Chemother. 45(5):1417-1421. and Example 1), 16-0.015 μg/mL;linezolid (ChemPacifica Inc., #35710), 64-0.06 μg/mL; Daptomycin (CubistPharmaceuticals #MCB2007), 32-0.03 μg/mL and 16-0.015 μg/mL; vancomycin(Sigma-Aldrich, St. Louis, Mo., #V2002), 64-0.06 μg/mL; ceftazidime,(Sigma #C3809), 64-0.06 μg/mL and 32-0.03 μg/mL; imipenem (United StatesPharmacopeia, N.J., #1337809) 16-0.015 μg/mL and 8-0.008 μg/mL;ciprofloxacin (United States Pharmacopeia, #IOC265), 32-0.03 μg/mL and4-0.004 μg/mL; gentamicin (Sigma #G3632) 32-0.03 μg/mL and 16-0.015μg/mL. All test articles, except gentamicin, were dissolved in DMSO;gentamicin was dissolved in water. Stock solutions were prepared at40-fold the highest concentration in the test plate. The finalconcentration of DMSO in the test system was 2.5%.

Organisms. The test organisms were obtained from clinical laboratoriesas follows: CHP, Clarian Health Partners, Indianapolis, Ind.; UCLA,University of California Los Angeles Medical Center, Los Angeles,Calif.; GR Micro, London, UK; PHRI TB Center, Public Health ResearchInstitute Tuberculosis Center, New York, N.Y.; ATCC, American TypeCulture Collection, Manassas, Va.; Mt Sinai Hosp., Mount Sinai Hospital,New York, N.Y.; UCSF, University of California San Francisco GeneralHospital, San Francisco, Calif.; Bronson Hospital, Bronson MethodistHospital, Kalamazoo, Mich.; quality control isolates were from theAmerican Type Culture Collection (ATCC, Manassas, Va.). Organisms werestreaked for isolation on agar medium appropriate to each organism.Colonies were picked by swab from the isolation plates and put intosuspension in appropriate broth containing a cryoprotectant. Thesuspensions were aliquoted into cryogenic vials and maintained at −80°C. Abbreviations are: BisEDT, bismuth-1,2-ethanedithiol; LZD, linezolid;DAP, daptomycin; VA, vancomycin; CAZ, ceftazidime; IPM, imipenem; CIP,ciprofloxacin; GM, gentamicin; MSSA, methicillin-susceptibleStaphylococcus aureus; CLSI QC, Clinical and Laboratory StandardsInstitute quality control strain; MRSA, methicillin-resistantStaphylococcus aureus; CA-MRSA, community-acquired methicillin-resistantStaphylococcus aureus; MSSE, methicillin-susceptible Staphylococcusepidermidis; MRSE, methicillin-resistant Staphylococcus epidermidis;VSE, vancomycin-susceptible Enterococcus.

The isolates were streaked from the frozen vials onto appropriatemedium: Trypticase Soy Agar (Becton-Dickinson, Sparks, Md.) for mostorganisms or Trypticase Soy Agar plus 5% sheep blood (ClevelandScientific, Bath, Ohio) for streptococci. The plates were incubatedovernight at 35° C. Quality control organisms were included. The mediumemployed for the MIC assay was Mueller Hinton II Broth (MHB II—BectonDickinson, #212322) for most of the organisms. MHB II was supplementedwith 2% lysed horse blood (Cleveland Scientific Lot #H13913) toaccommodate the growth of Streptococcus pyogenes and Streptococcusagalactiae. The media were prepared at 102.5% normal weight to offsetthe dilution created by the addition of 5 μL drug solution to each wellof the microdilution panels. In addition, for tests with daptomycin, themedium was supplemented with an additional 25 mg/L Ca²⁺.

The MIC assay method followed the procedure described by the Clinicaland Laboratory Standards Institute (Clinical and Laboratory StandardsInstitute. Methods for Dilution Antimicrobial Susceptibility Tests forBacteria That Grow Aerobically; Approved Standard—Seventh Edition.Clinical and Laboratory Standards Institute document M7-A7 [ISBN1-56238-587-9]. Clinical and Laboratory Standards Institute, 940 WestValley Road, Suite 1400, Wayne, Pa. 19087-1898 USA, 2006) and employedautomated liquid handlers to conduct serial dilutions and liquidtransfers. Automated liquid handlers included the Multidrop 384(Labsystems, Helsinki, Finland), Biomek 2000 and Multimek 96 (BeckmanCoulter, Fullerton Calif.). The wells of Columns 2-12 of standard96-well microdilution plates (Falcon 3918) were filled with 150 μL ofDMSO or water for gentamicin on the Multidrop 384. The drugs (300 μL)were dispensed into Column 1 of the appropriate row in these plates.These would become the mother plates from which the test plates(daughter plates) were prepared. The Biomek 2000 completed serialtransfers through Column 11 in the mother plates. The wells of Column 12contained no drug and were the organism growth control wells in thedaughter plates. The daughter plates were loaded with 185 μL of theappropriate test media (described above) using the Multidrop 384. Thedaughter plates were prepared on the Multimek 96 instrument whichtransferred 5 μL of drug solution from each well of a mother plate toeach corresponding well of each daughter plate in a single step.

Standardized inoculum of each organism was prepared per CLSI methods(ISBN 1-56238-587-9, cited supra). Suspensions were prepared in MHB toequal the turbidity of a 0.5 McFarland standard. The suspensions werediluted 1:9 in broth appropriate to the organism. The inoculum for eachorganism was dispensed into sterile reservoirs divided by length(Beckman Coulter), and the Biomek 2000 was used to inoculate the plates.Daughter plates were placed on the Biomek 2000 work surface reversed sothat inoculation took place from low to high drug concentration. TheBiomek 2000 delivered 10 μL of standardized inoculum into each well.This yielded a final cell concentration in the daughter plates ofapproximately 5×105 colony-forming-units/mL. Thus, the wells of thedaughter plates ultimately contained 185 μL of broth, 5 μL of drugsolution, and 10 μL of bacterial inoculum. Plates were stacked 3 high,covered with a lid on the top plate, placed in plastic bags, andincubated at 35° C. for approximately 18 hours for most of the isolates.The Streptococcus plates were read after 20 hours incubation. Themicroplates were viewed from the bottom using a plate viewer. For eachof the test media, an uninoculated solubility control plate was observedfor evidence of drug precipitation. The MIC was read and recorded as thelowest concentration of drug that inhibited visible growth of theorganism.

Results. All marketed drugs were soluble at all of the testconcentrations in both media. BisEDT exhibited a trace precipitate at 32μg/mL, but MIC readings were not affected as the inhibitoryconcentrations for all organisms tested were well below thatconcentration. On each assay day, an appropriate quality controlstrain(s) was included in the MIC assays. The MIC values derived forthese strains were compared to the published quality control ranges(Clinical and Laboratory Standards Institute. Performance Standards forAntimicrobial Susceptibility Testing; Eighteenth InformationalSupplement. CLSI document M100-S18 [ISBN 1-56238-653-0]. Clinical andLaboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne,Pa. 19087-1898 USA, 2008) for each agent, as appropriate.

On each assay day, an appropriate quality control strain(s) was includedin the MIC assays. The MIC values derived for these strains werecompared to the published quality control ranges (Clinical andLaboratory Standards Institute. Performance Standards for AntimicrobialSusceptibility Testing; Eighteenth Informational Supplement. CLSIdocument M100-S18 [ISBN 1-56238-653-0]) for each agent, as appropriate.Of 141 values for quality control strains where quality control rangesare published, 140(99.3%) were within the specified ranges. The oneexception was imipenem versus S. aureus 29213 which yielded one value ona single run (≤0.008 μg/mL) that was one dilution below the published QCrange. All other quality control results on that run were within thespecified quality control ranges.

BisEDT demonstrated potent activity against both methicillin-susceptibleStaphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA),and community-acquired MRSA (CA-MRSA), inhibiting all strains tested at1 μg/mL or less with an MIC90 values of 0.5 μg/mL for all three organismgroups. BisEDT exhibited activity greater than that of linezolid andvancomycin and equivalent to that of daptomycin. Imipenem was morepotent than BisEDT against MSSA (MIC90=0.03 μg/mL). However, MRSA andCAMRSA were resistant to imipenem while BisEDT demonstrated activityequivalent to that shown for MSSA. BisEDT was highly-active againstmethicillin-susceptible and methicillin-resistant Staphylococcusepidermidis (MSSE and MRSE), with MIC90 values of 0.12 and 0.25 μg/mL,respectively. BisEDT was more active against MSSE than any of the otheragents tested except imipenem. BisEDT was the most active agent testedagainst MRSE.

BisEDT demonstrated activity equivalent to that of daptomycin,vancomycin, and imipenem against vancomycin-susceptible Enterococcusfaecalis (VSEfc) with an MIC90 value of 2 μg/mL. Significantly, BisEDTwas the most active agent tested against vancomycin-resistantEnterococcus faecalis (VREfc) with an MIC90 value of 1 μg/mL.

BisEDT was very active against vancomycin-susceptible Enterococcusfaecium (VSEfm) with an MIC90 value of 2 μg/mL; its activity wasequivalent to that or similar to that of daptomycin and one-dilutionhigher than that of vancomycin. BisEDT and linezolid were the mostactive agents tested against vancomycin-resistant Enterococcus faecium(VREfm), each demonstrating an MIC90 value of 2 μg/mL. The activity ofBisEDT against Streptococcus pyogenes (MIC90 value of 0.5 μg/mL) wasequivalent to that of vancomycin, greater than that of linezolid, andslightly less than that of daptomycin and ceftazidime. The compoundinhibited all strains tested at 0.5 μg/mL or less. In these studies, thespecies that was least sensitive to BisEDT was Streptococcus agalactiaewhere the observed MIC90 value was 16 μg/mL. BisEDT was less active thanall of the agents tested except gentamicin.

The activity of BisEDT and comparators against Gram-negative bacteriaincluded demonstrated BisEDT potency against Acinetobacter baumanii(MIC90 value of 2 μg/mL) making BisEDT the most active compound tested.Elevated MICs for a significant number of test isolates for thecomparator agents resulted in off-scale MIC90 values for these agents.BisEDT was a potent inhibitor of Escherichia coli, inhibiting allstrains at 2 μg/mL or less (MIC90=2 μg/mL). The compound was less activethan imipenem, but more active than ceftazidime, ciprofloxacin, andgentamicin. BisEDT also demonstrated activity against Klebsiellapneumoniae with an MIC90 value of 8 μg/mL which was equivalent to thatof imipenem. The relatively high MIC90 values exhibited by imipenem,ceftazidime, ciprofloxacin, and gentamicin indicated that this was ahighly antibiotic-resistant group of organisms. BisEDT was the mostactive compound tested against Pseudomonas aeruginosa with an MIC90value of 4 μg/mL. There was a high level of resistance to the comparatoragents for this group of test isolates.

In summary, BisEDT demonstrated broad-spectrum potency against multipleclinical isolates representing multiple species, including speciescommonly involved in acute and chronic skin and skin structureinfections in humans. The activity of BisEDT and key comparator agentswas evaluated against 723 clinical isolates of Gram-positive andGram-negative bacteria. The BT compound demonstrated broad spectrumactivity, and for a number of the test organisms in this study, BisEDTwas the most active compound tested in terms of anti-bacterial activity.BisEDT was most active against MSSA, MRSA, CA-MRSA, MSSE, MRSE, and S.pyogenes, where the MIC90 value was 0.5 μg/mL or less. Potent activitywas also demonstrated for VSEfc, VREfc, VSEfm, VREfm, A. baumanii, E.coli, and P. aeruginosa where the MIC90 value was in the range of 1-4μg/mL. MIC90 values observed were, for K. pneumoniae (MIC90=8 μg/mL),and for S. agalactiae (MIC90=16 μg/mL).

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

1.-63. (canceled)
 64. A bismuth-thiol (BT) composition, comprising aplurality of microparticles, wherein at least 80% of the microparticleshave a volumetric mean diameter (VMD) of from about 0.4 μm to about 5μm, said microparticles comprising BisEDT.
 65. The composition of claim64, wherein the composition further comprises a pharmaceuticallyacceptable carrier.
 66. The composition of claim 64, wherein at least80% of the microparticles have a VMD of 1.0 μm to 3 μm.
 67. Thecomposition of claim 64, wherein at least 85% of all of themicroparticles have a VMD of 0.4 μm to 5 μm.
 68. The composition ofclaim 64, wherein at least 95% of all of the microparticles have a VMDof 0.4 μm to 5 μm.
 69. The composition of claim 64, wherein themicroparticles have a peak VMD of about 1.3 microns.
 70. The compositionof claim 64, wherein the size distribution of said microparticles isunimodal.
 71. The composition of claim 67, wherein the size distributionof said microparticles is unimodal.
 72. The composition of claim 64,wherein the composition further comprises methylcellulose, sodiumchloride and Tween®.
 73. The composition of claim 64, wherein thecomposition is a liquid suspension.
 74. The composition of claim 64,wherein the composition is an aerosol suspension.
 75. The composition ofclaim 64, wherein the microparticles are solid microparticles.
 76. Thecomposition of claim 64, wherein the composition does not include aliposome.